Humanized antibodies that recognize beta amyloid peptide

ABSTRACT

The invention provides improved agents and methods for treatment of diseases associated with amyloid deposits of Aβ in the brain of a patient. Preferred agents include humanized antibodies.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/396,417, filed Mar. 30, 2006, which is a continuation of U.S.application Ser. No. 10/704,070, filed Nov. 7, 2003. This application isalso a continuation of U.S. application Ser. No. 10/704,070, filed Nov.7, 2003.

This application is also a continuation-in-part of U.S. application Ser.No. 10/232,030, filed Aug. 30, 2002, which is a continuation of U.S.application Ser. No. 10/010,942, filed Dec. 6, 2001, which claims thebenefit under 35 U.S.C. 119(e) of U.S. Application No. 60/251,892, filedDec. 6, 2000.

This application is also a continuation-in-part of U.S. application Ser.No. 11/058,757, filed Feb. 14, 2005, which is a continuation of U.S.application Ser. No. 09/724,940, filed Nov. 28, 2000 (now U.S. Pat. No.6,905,686), which is a continuation of U.S. application Ser. No.09/580,015, filed May 26, 2000 (now abandoned), which is acontinuation-in-part of U.S. application Ser. No. 09/322,289, filed May28, 1999, which is a continuation-in-part of U.S. application Ser. No.09/201,430, filed Nov. 30, 1998 (now U.S. Pat. No. 6,787,523), whichclaims the benefit under 35 U.S.C. 119(e) of U.S. Application No.60/080,970, filed Apr. 7, 1998. Each of the above-referencedapplications is incorporated herein by reference for all purposes.

BACKGROUND OF THE INVENTION

Alzheimer's disease-(AD) is a progressive disease resulting in seniledementia. See generally Selkoe, TINS 16:403 (1993); Hardy et al., WO92/13069; Selkoe, J. Neuropathol. Exp. Neurol. 53:438 (1994); Duff etal., Nature 373:476 (1995); Games et al., Nature 373:523 (1995). Broadlyspeaking, the disease falls into two categories: late onset, whichoccurs in old age (65+ years) and early onset, which develops wellbefore the senile period, i.e., between 35 and 60 years. In both typesof disease, the pathology is the same but the abnormalities tend to bemore severe and widespread in cases beginning at an earlier age. Thedisease is characterized by at least two types of lesions in the brain,neurofibrillary tangles and senile plaques.

Neurofibrillary tangles are intracellular deposits of microtubuleassociated tau protein consisting of two filaments twisted about eachother in pairs. Senile plaques (i.e., amyloid plaques) are areas ofdisorganized neuropil up to 150 μm across with extracellular amyloiddeposits at the center which are visible by microscopic analysis ofsections of brain tissue. The accumulation of amyloid plaques within thebrain is also associated with Down's syndrome and other cognitivedisorders.

The principal constituent of the plaques is a peptide termed Aβ orβ-amyloid peptide. Aβ peptide is a 4-kDa internal fragment of 39-43amino acids of a larger transmembrane glycoprotein named protein termedamyloid precursor protein (APP). As a result of proteolytic processingof APP by different secretase enzymes, Aβ is primarily found in both ashort form, 40 amino acids in length, and a long form, ranging from42-43 amino acids in length. Part of the hydrophobic transmembranedomain of APP is found at the carboxy end of Aβ, and may account for theability of Aβ to aggregate into plaques, particularly in the case of thelong form. Accumulation of amyloid plaques in the brain eventually leadsto neuronal cell death. The physical symptoms associated with this typeof neural deterioration characterize Alzheimer's disease.

Several mutations within the APP protein have been correlated with thepresence of Alzheimer's disease. See, e.g., Goate et al., Nature349:704) (1991) (valine⁷¹⁷ to isoleucine); Chartier Harlan et al. Nature353:844 (1991)) (valine⁷¹⁷ to glycine); Murrell et al., Science 254:97(1991) (valine⁷¹⁷ to phenylalanine); Mullan et al., Nature Genet. 1:345(1992) (a double mutation changing lysine⁵⁹⁵-methionine⁵⁹⁶ toasparagine⁵⁹⁵-leucine⁵⁹⁶). Such mutations are thought to causeAlzheimer's disease by increased or altered processing of APP to Aβ,particularly processing of APP to increased amounts of the long form ofAβ (i.e., Aβ1-42 and Aβ1-43). Mutations in other genes, such as thepresenilin genes, PS1 and PS2, are thought indirectly to affectprocessing of APP to generate increased amounts of long form Aβ (seeHardy, TINS 20: 154 (1997)).

Mouse models have been used successfully to determine the significanceof amyloid plaques in Alzheimer's (Games et al., supra, Johnson-Wood etal., Proc. Natl. Acad. Sci. USA 94:1550 (1997)). In particular, whenPDAPP transgenic mice, (which express a mutant form of human APP anddevelop Alzheimer's disease at a young age), are injected with the longform of Aβ, they display both a decrease in the progression ofAlzheimer's and an increase in antibody titers to the Aβ peptide (Schenket al., Nature 400, 173 (1999)). The observations discussed aboveindicate that Aβ, particularly in its long form, is a causative elementin Alzheimer's disease.

McMichael, EP 526,511, proposes administration of homeopathic dosages(less than or equal to 10⁻² mg/day) of Aβ to patients withpreestablished AD. In a typical human with about 5 liters of plasma,even the upper limit of this dosage would be expected to generate aconcentration of no more than 2 pg/ml. The normal concentration of Aβ inhuman plasma is typically in the range of 50-200 pg/ml (Seubert et al.,Nature 359:325 (1992)). Because EP 526,511's proposed dosage wouldbarely alter the level of endogenous circulating Aβ and because EP526,511 does not recommend use of an adjuvant, as an immunostimulant, itseems implausible that any therapeutic benefit would result.

Accordingly, there exists the need for new therapies and reagents forthe treatment of Alzheimer's disease, in particular, therapies andreagents capable of effecting a therapeutic benefit at physiologic(e.g., non-toxic) doses.

SUMMARY OF THE INVENTION

The present invention features new immunological reagents, inparticular, therapeutic antibody reagents for the prevention andtreatment of amyloidogenic disease (e.g., Alzheimer's disease). Theinvention is based, at least in part, on the identification andcharacterization of two monoclonal antibodies that specifically bind toAβ peptide and are effective at reducing plaque burden and/or reducingthe neuritic dystrophy associated with amyloidogenic disorders.Structural and functional analysis of these antibodies leads to thedesign of various humanized antibodies for prophylactic and/ortherapeutic use. In particular, the invention features humanization ofthe variable regions of these antibodies and, accordingly provides forhumanized immunoglobulin or antibody chains, intact humanizedimmunoglobulins or antibodies, and functional immunoglobulin or antibodyfragments, in particular, antigen binding fragments, of the featuredantibodies.

Polypeptides comprising the complementarity determining regions of thefeatured monoclonal antibodies are also disclosed, as are polynucleotidereagents, vectors and host suitable for encoding said polypeptides.

Methods of treatment of amyloidogenic diseases or disorders (e.g.,Alzheimer's disease) are disclosed, as are pharmaceutical compositionsand kits for use in such applications.

Also featured are methods of identifying residues within the featuredmonoclonal antibodies which are important for proper immunologicfunction and for identifying residues which are amenable to substitutionin the design of humanized antibodies having improved binding affinitiesand/or reduced immunogenicity, when used as therapeutic reagents.

Also featured are antibodies (e.g., humanized antibodies) having alteredeffector functions, and therapeutic uses thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an alignment of the amino acid sequences of the lightchain of mouse 3D6 (amino acids 1-112 of SEQ ID NO:2), humanized 3D6(amino acids 1-112 of SEQ ID NO:5), Kabat ID 109230 (amino acids 1-112of SEQ ID NO:6) and germline A19 antibodies (SEQ ID NO:7). CDR regionsare indicated by arrows. Bold italics indicate rare murine residues.Bold indicates packing (VH+VL) residues. Solid fill indicatescanonical/CDR interacting residues. Asterisks indicate residues selectedfor backmutation in humanized 3D6, version 1.

FIG. 2 depicts an alignment of the amino acid sequences of the heavychain of mouse 3D6 (amino acids 1-119 of SEQ ID NO:4), humanized 3D6(amino acids 1-119 of SEQ ID NO:8), Kabat ID 045919 (SEQ ID NO:9) andgermline VH3-23 antibodies (SEQ ID NO:10). Annotation is the same as forFIG. 1.

FIG. 3 graphically depicts the Aβ binding properties of 3D6, chimeric3D6 and 10D5. FIG. 3A is a graph depicting binding of Aβ to chimeric 3D6(PK1614) as compared to murine 3D6. FIG. 3B is a graph depictingcompetition of biotinylated 3D6 versus unlabeled 3D6, PK1614 and 10D5for binding to Aβ.

FIG. 4 depicts a homology model of 3D6 VH and VL, showing α-carbonbackbone trace. VH is shown in as a stippled line, and VL is shown as asolid line. CDR regions are indicated in ribbon form.

FIG. 5 graphically depicts the Aβ binding properties of chimeric 3D6 andhumanized 3D6. FIG. 5A depicts ELISA results measuring the binding ofhumanized 3D6v1 and chimeric 3D6 to aggregated Aβ. FIG. 5B depicts ELISAresults measuring the binding of humanized 3D6v1 and humanized 3D6v2 toaggregated Aβ.

FIG. 6 is a graph quantitating the binding of humanized 3D6 and chimeric3D6 to Aβ plaques from brain sections of PDAPP mice.

FIG. 7 is a graph showing results of a competitive binding assay testingthe ability of humanized 3D6 versions 1 and 2, chimeric 3D6, murine 3D6,and 10D5 to compete with murine 3D6 for binding to Aβ.

FIG. 8 graphically depicts of an ex vivo phagocytosis assay testing theability of humanized 3D6v2, chimeric 3D6, and human IgG to mediate theuptake of Aβ by microglial cells.

FIG. 9 depicts an alignment of the 10D5 VL (SEQ ID NO:14) and 3D6 VL(SEQ ID NO:2) amino acid sequences. Bold indicates residues that match10D5 exactly.

FIG. 10 depicts an alignment of the 10D5 VH (SEQ ID NO:16) and 3D6 VH(SEQ ID NO:4) amino acid sequences. Bold indicates residues that match10D5 exactly.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features new immunological reagents and methodsfor preventing or treating Alzheimer's disease or other amyloidogenicdiseases. The invention is based, at least in part, on thecharacterization of two monoclonal immunoglobulins, 3D6 and 10D5,effective at binding beta amyloid protein (Aβ) (e.g., binding solubleand/or aggregated Aβ), mediating phagocytosis (e.g., of aggregated Aβ),reducing plaque burden and/or reducing neuritic dystrophy (e.g., inpatient). The invention is further based on the determination andstructural characterization of the primary and secondary structure ofthe variable light and heavy chains of these immunoglobulins and theidentification of residues important for activity and immunogenicity.

Immunoglobulins are featured which include a variable light and/orvariable heavy chain of the preferred monoclonal immunoglobulinsdescribed herein. Preferred immunoglobulins, e.g., therapeuticimmunoglobulins, are featured which include a humanized variable lightand/or humanized variable heavy chain. Preferred variable light and/orvariable heavy chains include a complementarity determining region (CDR)from the monoclonal immunoglobulin (e.g., donor immunoglobulin) andvariable framework regions substantially from a human acceptorimmunoglobulin. The phrase “substantially from a human acceptorimmunoglobulin” means that the majority or key framework residues arefrom the human acceptor sequence, allowing however, for substitution ofresidues at certain positions with residues selected to improve activityof the humanized immunoglobulin (e.g., alter activity such that it moreclosely mimics the activity of the donor immunoglobulin) or selected todecrease the immunogenicity of the humanized immunoglobulin.

In one embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 3D6 variable region complementaritydetermining regions (CDRs) (i.e., includes one, two or three CDRs fromthe light chain variable region sequence set forth as SEQ ID NO:2 orincludes one, two or three CDRs from the heavy chain variable regionsequence set forth as SEQ ID NO:4), and includes a variable frameworkregion substantially from a human acceptor immunoglobulin light or heavychain sequence, provided that at least one residue of the frameworkresidue is backmutated to a corresponding murine residue, wherein saidbackmutation does not substantially affect the ability of the chain todirect Aβ binding.

In another embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 3D6 variable region complementaritydetermining regions (CDRs) (e.g., includes one, two or three CDRs fromthe light chain variable region sequence set forth as SEQ ID NO:2 and/orincludes one, two or three CDRs from the heavy chain variable regionsequence set forth as SEQ ID NO:4), and includes a variable frameworkregion substantially from a human acceptor immunoglobulin light or heavychain sequence, provided that at least one framework residue issubstituted with the corresponding amino acid residue from the mouse 3D6light or heavy chain variable region sequence, where the frameworkresidue is selected from the group consisting of (a) a residue thatnon-covalently binds antigen directly; (b) a residue adjacent to a CDR;(c) a CDR-interacting residue (e.g., identified by modeling the light orheavy chain on the solved structure of a homologous known immunoglobulinchain); and (d) a residue participating in the VL−VH interface.

In another embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 3D6 variable region CDRs and variableframework regions from a human acceptor immunoglobulin light or heavychain sequence, provided that at least one framework residue issubstituted with the corresponding amino acid residue from the mouse 3D6light or heavy chain variable region sequence, where the frameworkresidue is a residue capable of affecting light chain variable regionconformation or function as identified by analysis of athree-dimensional model of the variable region, for example a residuecapable of interacting with antigen, a residue proximal to the antigenbinding site, a residue capable of interacting with a CDR, a residueadjacent to a CDR, a residue within 6 Å of a CDR residue, a canonicalresidue, a vernier zone residue, an interchain packing residue, anunusual residue, or a glycoslyation site residue on the surface of thestructural model.

In another embodiment, the invention features a humanized immunoglobulinlight chain that includes 3D6 variable region CDRs (e.g., from the 3D6light chain variable region sequence set forth as SEQ ID NO:2), andincludes a human acceptor immunoglobulin variable framework region,provided that at least one framework residue selected from the groupconsisting of L1, L2, L36 and L46 (Kabat numbering convention) issubstituted with the corresponding amino acid residue from the mouse 3D6light chain variable region sequence. In another embodiment, theinvention features a humanized immunoglobulin heavy chain that includes3D6 variable region CDRs (e.g., from the 3D6 heavy chain variable regionsequence set forth as SEQ ID NO:4), and includes a human acceptorimmunoglobulin variable framework region, provided that at least oneframework residue selected from the group consisting of H49, H93 and H94(Kabat numbering convention) is substituted with the corresponding aminoacid residue from the mouse 3D6 heavy chain variable region sequence.

Preferred light chains include kappa II framework regions of the subtypekappa II (Kabat convention), for example, framework regions from theacceptor immunoglobulin Kabat ID 019230, Kabat ID 005131, Kabat ID005058, Kabat ID 005057, Kabat ID 005059, Kabat ID U21040 and Kabat IDU41645. Preferred heavy chains include framework regions of the subtypeIII (Kabat convention), for example, framework regions from the acceptorimmunoglobulin Kabat ID 045919, Kabat ID 000459, Kabat ID 000553, KabatID 000386 and Kabat ID M23691.

In one embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 10D5 variable region complementaritydetermining regions (CDRs) (i.e., includes one, two or three CDRs fromthe light chain variable region sequence set forth as SEQ ID NO:14 orincludes one, two or three CDRs from the heavy chain variable regionsequence set forth as SEQ ID NO:16), and includes a variable frameworkregion substantially from a human acceptor immunoglobulin light or heavychain sequence, provided that at least one residue of the frameworkresidue is backmutated to a corresponding murine residue, wherein saidbackmutation does not substantially affect the ability of the chain todirect Aβ binding.

In another embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 10D5 variable region complementaritydetermining regions (CDRs) (e.g., includes one, two or three CDRs fromthe light chain variable region sequence set forth as SEQ ID NO:14and/or includes one, two or three CDRs from the heavy chain variableregion sequence set forth as SEQ ID NO:16), and includes a variableframework region substantially from a human acceptor immunoglobulinlight or heavy chain sequence, provided that at least one frameworkresidue is substituted with the corresponding amino acid residue fromthe mouse 3D6 light or heavy chain variable region sequence, where theframework residue is selected from the group consisting of (a) a residuethat non-covalently binds antigen directly; (b) a residue adjacent to aCDR; (c) a CDR-interacting residue (e.g., identified by modeling thelight or heavy chain on the solved structure of a homologous knownimmunoglobulin chain); and (d) a residue participating in the VL−VHinterface.

In another embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 10D5 variable region CDRs andvariable framework regions from a human acceptor immunoglobulin light orheavy chain sequence, provided that at least one framework residue issubstituted with the corresponding amino acid residue from the mouse10D5 light or heavy chain variable region sequence, where the frameworkresidue is a residue capable of affecting light chain variable regionconformation or function as identified by analysis of athree-dimensional model of the variable region, for example a residuecapable of interacting with antigen, a residue proximal to the antigenbinding site, a residue capable of interacting with a CDR, a residueadjacent to a CDR, a residue within 6 Å of a CDR residue, a canonicalresidue, a vernier zone residue, an interchain packing residue, anunusual residue, or a glycoslyation site residue on the surface of thestructural model.

In another embodiment, the invention features, in addition to thesubstitutions described above, a substitution of at least one rare humanframework residue. For example, a rare residue can be substituted withan amino acid residue which is common for human variable chain sequencesat that position. Alternatively, a rare residue can be substituted witha corresponding amino acid residue from a homologous germline variablechain sequence (e.g., a rare light chain framework residue can besubstituted with a corresponding germline residue from an A1, A17, A18,A2, or A19 germline immunoglobulin sequence or a rare heavy chainframework residue can be substituted with a corresponding germlineresidue from a VH3-48, VH3-23, VH3-7, VH3-21 or VH3-11 germlineimmunoglobulin sequence.

In another embodiment, the invention features a humanized immunoglobulinthat includes a light chain and a heavy chain, as described above, or anantigen-binding fragment of said immunoglobulin. In an exemplaryembodiment, the humanized immunoglobulin binds (e.g., specificallybinds) to beta amyloid peptide (Aβ) with a binding affinity of at least10⁷ M⁻¹, 10⁸ M⁻¹, or 10⁹ M⁻¹. In another embodiment, the immunoglobulinor antigen binding fragment includes a heavy chain having isotype γ1. Inanother embodiment, the immunoglobulin or antigen binding fragment binds(e.g., specifically binds) to both soluble beta amyloid peptide (Aβ) andaggregated Aβ. In another embodiment, the immunoglobulin or antigenbinding fragment mediates phagocytosis (e.g., induces phagocytosis) ofbeta amyloid peptide (Aβ). In yet another embodiment, the immunoglobulinor antigen binding fragment crosses the blood-brain barrier in asubject. In yet another embodiment, the immunoglobulin or antigenbinding fragment reduces both beta amyloid peptide (Aβ) burden andneuritic dystrophy in a subject.

In another embodiment, the invention features chimeric immunoglobulinsthat include 3D6 variable regions (e.g., the variable region sequencesset forth as SEQ ID NO:2 or SEQ ID NO:4). As used herein, an antibody orimmunoglobulin sequence comprising a VL and/or VH sequence as set forthin, for example, SEQ ID NO:2 or SEQ ID NO:4 can comprise either the fullVL or VH sequence or can comprise the mature VL or VH sequence (i.e.,mature peptide without the signal or leader peptide). In yet anotherembodiment, the invention features an immunoglobulin, or antigen-bindingfragment thereof, including a variable heavy chain region as set forthin SEQ ID NO:8 and a variable light chain region as set forth in SEQ IDNO:5. In yet another embodiment, the invention features animmunoglobulin, or antigen-binding fragment thereof, including avariable heavy chain region as set forth in SEQ ID NO:12 and a variablelight chain region as set forth in SEQ ID NO:11. In another embodiment,the invention features chimeric immunoglobulins that include 10D5variable regions (e.g., the variable region sequences set forth as SEQID NO:14 or SEQ ID NO:16). In yet another embodiment, theimmunoglobulin, or antigen-binding fragment thereof, further includesconstant regions from IgG1.

The immunoglobulins described herein are particularly suited for use intherapeutic methods aimed at preventing or treating amyloidogenicdiseases. In one embodiment, the invention features a method ofpreventing or treating an amyloidogenic disease (e.g., Alzheimer'sdisease) that involves administering to the patient an effective dosageof a humanized immunoglobulin as described herein. In anotherembodiment, the invention features pharmaceutical compositions thatinclude a humanized immunoglobulin as described herein and apharmaceutical carrier. Also featured are isolated nucleic acidmolecules, vectors and host cells for producing the immunoglobulins orimmunoglobulin fragments or chains described herein, as well as methodsfor producing said immunoglobulins, immunoglobulin fragments orimmunoglobulin chains.

The present invention further features a method for identifying 3D6 or10D5 residues amenable to substitution when producing a humanized 3D6 or10D5 immunoglobulin, respectively. For example, a method for identifyingvariable framework region residues amenable to substitution involvesmodeling the three-dimensional structure of the 3D6 or 10D5 variableregion on a solved homologous immunoglobulin structure and analyzingsaid model for residues capable of affecting 3D6 or 10D5 immunoglobulinvariable region conformation or function, such that residues amenable tosubstitution are identified. The invention further features use of thevariable region sequence set forth as SEQ ID NO:2 or SEQ ID NO:4, or anyportion thereof, in producing a three-dimensional image of a 3D6immunoglobulin, 3D6 immunoglobulin chain, or domain thereof. Alsofeatured is the use of the variable region sequence set forth as SEQ IDNO:14 or SEQ ID NO:16, or any portion thereof, in producing athree-dimensional image of a 10D5 immunoglobulin, 10D5 immunoglobulinchain, or domain thereof.

The present invention further features immunoglobulins having alteredeffector function, such as the ability to bind effector molecules, forexample, complement or a receptor on an effector cell. In particular,the immunoglobulin of the invention has an altered constant region,e.g., Fc region, wherein at least one amino acid residue in the Fcregion has been replaced with a different residue or side chain. In oneembodiment, the modified immunoglobulin is of the IgG class, comprisesat least one amino acid residue replacement in the Fc region such thatthe immunoglobulin has an altered effector function, e.g., as comparedwith an unmodified immunoglobulin. In particular embodiments, theimmunoglobulin of the invention has an altered effector function suchthat it is less immunogenic (e.g., does not provoke undesired effectorcell activity, lysis, or complement binding), has improved amyloidclearance properties, and/or has a desirable half-life.

Prior to describing the invention, it may be helpful to an understandingthereof to set forth definitions of certain terms to be usedhereinafter.

The term “immunoglobulin” or “antibody” (used interchangeably herein)refers to an antigen-binding protein having a basic four-polypeptidechain structure consisting of two heavy and two light chains, saidchains being stabilized, for example, by interchain disulfide bonds,which has the ability to specifically bind antigen. Both heavy and lightchains are folded into domains. The term “domain” refers to a globularregion of a heavy or light chain polypeptide comprising peptide loops(e.g., comprising 3 to 4 peptide loops) stabilized, for example, byβ-pleated sheet and/or intrachain disulfide bond. Domains are furtherreferred to herein as “constant” or “variable”, based on the relativelack of sequence variation within the domains of various class membersin the case of a “constant” domain, or the significant variation withinthe domains of various class members in the case of a “variable” domain.“Constant” domains on the light chain are referred to interchangeably as“light chain constant regions”, “light chain constant domains”, “CL”regions or “CL” domains). “Constant” domains on the heavy chain arereferred to interchangeably as “heavy chain constant regions”, “heavychain constant domains”, “CH” regions or “CH” domains). “Variable”domains on the light chain are referred to interchangeably as “lightchain variable regions”, “light chain variable domains”, “VL” regions or“VL” domains). “Variable” domains on the heavy chain are referred tointerchangeably as “heavy chain constant regions”, “heavy chain constantdomains”, “CH” regions or “CH” domains).

The term “region” refers to a part or portion of an antibody chain andincludes constant or variable domains as defined herein, as well as morediscrete parts or portions of said domains. For example, light chainvariable domains or regions include “complementarity determiningregions” or “CDRs” interspersed among “framework regions” or “FRs”, asdefined herein.

Immunoglobulins or antibodies can exist in monomeric or polymeric form.The term “antigen-binding fragment” refers to a polypeptide fragment ofan immunoglobulin or antibody binds antigen or competes with intactantibody (i.e., with the intact antibody from which they were derived)for antigen binding (i.e., specific binding). The term “conformation”refers to the tertiary structure of a protein or polypeptide (e.g., anantibody, antibody chain, domain or region thereof). For example, thephrase “light (or heavy) chain conformation” refers to the tertiarystructure of a light (or heavy) chain variable region, and the phrase“antibody conformation” or “antibody fragment conformation” refers tothe tertiary structure of an antibody or fragment thereof.

“Specific binding” of an antibody mean that the antibody exhibitsappreciable affinity for antigen or a preferred epitope and, preferably,does not exhibit significant crossreactivity. “Appreciable” or preferredbinding include binding with an affinity of at least 10⁶, 10⁷, 10⁸, 10⁹M⁻¹, or 10¹⁰ M⁻¹. Affinities greater than 10⁷ M⁻¹, preferably greaterthan 10⁸ M⁻¹ are more preferred. Values intermediate of those set forthherein are also intended to be within the scope of the present inventionand a preferred binding affinity can be indicated as a range ofaffinities, for example, 10⁶ to 10¹⁰ M⁻¹, preferably 10⁷ to 10¹⁰ M⁻¹,more preferably 10⁸ to 10¹⁰ M⁻¹. An antibody that “does not exhibitsignificant crossreactivity” is one that will not appreciably bind to anundesirable entity (e.g., an undesirable proteinaceous entity). Forexample, an antibody that specifically binds to Aβ will appreciably bindAβ but will not significantly react with non-Aβ proteins or peptides(e.g., non-Aβ proteins or peptides included in plaques). An antibodyspecific for a preferred epitope will, for example, not significantlycrossreact with remote epitopes on the same protein or peptide. Specificbinding can be determined according to any art-recognized means fordetermining such binding. Preferably, specific binding is determinedaccording to Scatchard analysis and/or competitive binding assays.

Binding fragments are produced by recombinant DNA techniques, or byenzymatic or chemical cleavage of intact immunoglobulins. Bindingfragments include Fab, Fab′, F(ab′)₂, Fabc, Fv, single chains, andsingle-chain antibodies. Other than “bispecific” or “bifunctional”immunoglobulins or antibodies, an immunoglobulin or antibody isunderstood to have each of its binding sites identical. A “bispecific”or “bifunctional antibody” is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelnyet al., J. Immunol. 148, 1547-1553 (1992).

The term “humanized immunoglobulin” or “humanized antibody” refers to animmunoglobulin or antibody that includes at least one humanizedimmunoglobulin or antibody chain (i.e., at least one humanized light orheavy chain). The term “humanized immunoglobulin chain” or “humanizedantibody chain” (i.e., a “humanized immunoglobulin light chain” or“humanized immunoglobulin heavy chain”) refers to an immunoglobulin orantibody chain (i.e., a light or heavy chain, respectively) having avariable region that includes a variable framework region substantiallyfrom a human immunoglobulin or antibody and complementarity determiningregions (CDRs) (e.g., at least one CDR, preferably two CDRs, morepreferably three CDRs) substantially from a non-human immunoglobulin orantibody, and further includes constant regions (e.g., at least oneconstant region or portion thereof, in the case of a light chain, andpreferably three constant regions in the case of a heavy chain). Theterm “humanized variable region” (e.g., “humanized light chain variableregion” or “humanized heavy chain variable region”) refers to a variableregion that includes a variable framework region substantially from ahuman immunoglobulin or antibody and complementarity determining regions(CDRs) substantially from a non-human immunoglobulin or antibody.

The phrase “substantially from a human immunoglobulin or antibody” or“substantially human” means that, when aligned to a human immunoglobulinor antibody amino sequence for comparison purposes, the region shares atleast 80-90%, preferably 90-95%, more preferably 95-99% identity (i.e.,local sequence identity) with the human framework or constant regionsequence, allowing, for example, for conservative substitutions,consensus sequence substitutions, germline substitutions, backmutations,and the like. The introduction of conservative substitutions, consensussequence substitutions, germline substitutions, backmutations, and thelike, is often referred to as “optimization” of a humanized antibody orchain. The phrase “substantially from a non-human immunoglobulin orantibody” or “substantially non-human” means having an immunoglobulin orantibody sequence at least 80-95%, preferably 90-95%, more preferably,96%, 97%, 98%, or 99% identical to that of a non-human organism, e.g., anon-human mammal.

Accordingly, all regions or residues of a humanized immunoglobulin orantibody, or of a humanized immunoglobulin or antibody chain, exceptpossibly the CDRs, are substantially identical to the correspondingregions or residues of one or more native human immunoglobulinsequences. The term “corresponding region” or “corresponding residue”refers to a region or residue on a second amino acid or nucleotidesequence which occupies the same (i.e., equivalent) position as a regionor residue on a first amino acid or nucleotide sequence, when the firstand second sequences are optimally aligned for comparison purposes.

The terms “humanized immunoglobulin” or “humanized antibody” are notintended to encompass chimeric immunoglobulins or antibodies, as definedinfra. Although humanized immunoglobulins or antibodies are chimeric intheir construction (i.e., comprise regions from more than one species ofprotein), they include additional features (i.e., variable regionscomprising donor CDR residues and acceptor framework residues) not foundin chimeric immunoglobulins or antibodies, as defined herein.

The term “significant identity” means that two polypeptide sequences,when optimally aligned, such as by the programs GAP or BESTFIT usingdefault gap weights, share at least 50-60% sequence identity, preferably60-70% sequence identity, more preferably 70-80% sequence identity, morepreferably at least 80-90% identity, even more preferably at least90-95% identity, and even more preferably at least 95% sequence identityor more (e.g., 99% sequence identity or more). The term “substantialidentity” means that two polypeptide sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights, shareat least 80-90% sequence identity, preferably 90-95% sequence identity,and more preferably at least 95% sequence identity or more (e.g., 99%sequence identity or more). For sequence comparison, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters. Theterms “sequence identity” and “sequence identity” are usedinterchangeably herein.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., Current Protocols in Molecular Biology). One example ofalgorithm that is suitable for determining percent sequence identity andsequence similarity is the BLAST algorithm, which is described inAltschul et al., J. Mol. Biol. 215:403 (1990). Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (publicly accessible through the NationalInstitutes of Health NCBI internet server). Typically, default programparameters can be used to perform the sequence comparison, althoughcustomized parameters can also be used. For amino acid sequences, theBLASTP program uses as defaults a wordlength (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff,Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

Preferably, residue positions which are not identical differ byconservative amino acid substitutions. For purposes of classifying aminoacids substitutions as conservative or nonconservative, amino acids aregrouped as follows: Group I (hydrophobic sidechains): leu, met, ala,val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser,thr; Group III (acidic side chains): asp, glu; Group IV (basic sidechains): asn, gln, his, lys, arg; Group V (residues influencing chainorientation): gly, pro; and Group VI (aromatic side chains): trp, tyr,phe. Conservative substitutions involve substitutions between aminoacids in the same class. Non-conservative substitutions constituteexchanging a member of one of these classes for a member of another.

Preferably, humanized immunoglobulins or antibodies bind antigen with anaffinity that is within a factor of three, four, or five of that of thecorresponding non-human antibody. For example, if the nonhuman antibodyhas a binding affinity of 10⁹ M⁻¹, humanized antibodies will have abinding affinity of at least 3×10⁹ M⁻¹, 4×10⁹ M⁻¹ or 10⁹ M⁻¹. Whendescribing the binding properties of an immunoglobulin or antibodychain, the chain can be described based on its ability to “directantigen (e.g., Aβ) binding”. A chain is said to “direct antigen binding”when it confers upon an intact immunoglobulin or antibody (or antigenbinding fragment thereof) a specific binding property or bindingaffinity. A mutation (e.g., a backmutation) is said to substantiallyaffect the ability of a heavy or light chain to direct antigen bindingif it affects (e.g., decreases) the binding affinity of an intactimmunoglobulin or antibody (or antigen binding fragment thereof)comprising said chain by at least an order of magnitude compared to thatof the antibody (or antigen binding fragment thereof) comprising anequivalent chain lacking said mutation. A mutation “does notsubstantially affect (e.g., decrease) the ability of a chain to directantigen binding” if it affects (e.g., decreases) the binding affinity ofan intact immunoglobulin or antibody (or antigen binding fragmentthereof) comprising said chain by only a factor of two, three, or fourof that of the antibody (or antigen binding fragment thereof) comprisingan equivalent chain lacking said mutation.

The term “chimeric immunoglobulin” or antibody refers to animmunoglobulin or antibody whose variable regions derive from a firstspecies and whose constant regions derive from a second species.Chimeric immunoglobulins or antibodies can be constructed, for exampleby genetic engineering, from immunoglobulin gene segments belonging todifferent species.

An “antigen” is an entity (e.g., a protenaceous entity or peptide) towhich an antibody specifically binds.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody (or antigen bindingfragment thereof) specifically binds. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

Antibodies that recognize the same epitope can be identified in a simpleimmunoassay showing the ability of one antibody to block the binding ofanother antibody to a target antigen, i.e., a competitive binding assay.Competitive binding is determined in an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as Aβ. Numerous types of competitivebinding assays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidinEIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress (1988)); solid phase direct label RIA using I-125 label (see Morelet al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidinEIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, suchan assay involves the use of purified antigen bound to a solid surfaceor cells bearing either of these, an unlabeled test immunoglobulin and alabeled reference immunoglobulin. Competitive inhibition is measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test immunoglobulin. Usually the test immunoglobulinis present in excess. Usually, when a competing antibody is present inexcess, it will inhibit specific binding of a reference antibody to acommon antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% ormore.

An epitope is also recognized by immunologic cells, for example, B cellsand/or T cells. Cellular recognition of an epitope can be determined byin vitro assays that measure antigen-dependent proliferation, asdetermined by ³H-thymidine incorporation, by cytokine secretion, byantibody secretion, or by antigen-dependent killing (cytotoxic Tlymphocyte assay).

Exemplary epitopes or antigenic determinants can be found within thehuman amyloid precursor protein (APP), but are preferably found withinthe Aβ peptide of APP. Multiple isoforms of APP exist, for exampleAPP⁶⁹⁵ APP⁷⁵¹ and APP⁷⁷⁰. Amino acids within APP are assigned numbersaccording to the sequence of the APP⁷⁷⁰ isoform (see e.g., GenBankAccession No. P05067, also set forth as SEQ ID NO:38). Aβ (also referredto herein as beta amyloid peptide and A-beta) peptide is a ˜4-kDainternal fragment of 39-43 amino acids of APP (Aβ39, Aβ40, Aβ41, Aβ42and Aβ43). Aβ40, for example, consists of residues 672-711 of APP andAβ42 consists of residues 673-713 of APP. As a result of proteolyticprocessing of APP by different secretase enzymes iv vivo or in situ, Aβis found in both a “short form”, 40 amino acids in length, and a “longform”, ranging from 42-43 amino acids in length. Preferred epitopes orantigenic determinants, as described herein, are located within theN-terminus of the Aβ peptide and include residues within amino acids1-10 of Aβ, preferably from residues 1-3, 1-4, 1-5, 1-6, 1-7 or 3-7 ofAβ42. Additional referred epitopes or antigenic determinants includeresidues 2-4, 5, 6, 7 or 8 of Aβ, residues 3-5, 6, 7, 8 or 9 of Aβ, orresidues 4-7, 8, 9 or 10 of Aβ42.

The term “amyloidogenic disease” includes any disease associated with(or caused by) the formation or deposition of insoluble amyloid fibrils.Exemplary amyloidogenic diseases include, but are not limited tosystemic amyloidosis, Alzheimer's disease, mature onset diabetes,Parkinson's disease, Huntington's disease, fronto-temporal dementia, andthe prion-related transmissible spongiform encephalopathies (kuru andCreutzfeldt-Jacob disease in humans and scrapie and BSE in sheep andcattle, respectively). Different amyloidogenic diseases are defined orcharacterized by the nature of the polypeptide component of the fibrilsdeposited. For example, in subjects or patients having Alzheimer'sdisease, β-amyloid protein (e.g., wild-type, variant, or truncatedβ-amyloid protein) is the characterizing polypeptide component of theamyloid deposit. Accordingly, Alzheimer's disease is an example of a“disease characterized by deposits of Aβ” or a “disease associated withdeposits of Aβ”, e.g., in the brain of a subject or patient. The terms“β-amyloid protein”, “β-amyloid peptide”, “β-amyloid”, “Aβ” and “Aβpeptide” are used interchangeably herein.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve the desired effect.The term “therapeutically effective dose” is defined as an amountsufficient to cure or at least partially arrest the disease and itscomplications in a patient already suffering from the disease. Amountseffective for this use will depend upon the severity of the infectionand the general state of the patient's own immune system.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

“Soluble” or “dissociated” Aβ refers to non-aggregating or disaggregatedAβ polypeptide. “Insoluble” Aβ refers to aggregating Aβ polypeptide, forexample, Aβ held together by noncovalent bonds. Aβ (e.g., Aβ42) isbelieved to aggregate, at least in part, due to the presence ofhydrophobic residues at the C-terminus of the peptide (part of thetransmembrane domain of APP). One method to prepare soluble Aβ is todissolve lyophilized peptide in neat DMSO with sonication. The resultingsolution is centrifuged to remove any insoluble particulates.

The term “effector function” refers to an activity that resides in theFc region of an antibody (e.g., an IgG antibody) and includes, forexample, the ability of the antibody to bind effector molecules such ascomplement and/or Fc receptors, which can control several immunefunctions of the antibody such as effector cell activity, lysis,complement-mediated activity, antibody clearance, and antibodyhalf-life.

The term “effector molecule” refers to a molecule that is capable ofbinding to the Fc region of an antibody (e.g., an IgG antibody)including, but not limited to, a complement protein or a Fc receptor.

The term “effector cell” refers to a cell capable of binding to the Fcportion of an antibody (e.g., an IgG antibody) typically via an Fcreceptor expressed on the surface of the effector cell including, butnot limited to, lymphocytes, e.g., antigen presenting cells and T cells.

The term “Fc region” refers to a C-terminal region of an IgG antibody,in particular, the C-terminal region of the heavy chain(s) of said IgGantibody. Although the boundaries of the Fc region of an IgG heavy chaincan vary slightly, a Fc region is typically defined as spanning fromabout amino acid residue Cys226 to the carboxyl-terminus of an IGg heavychain(s).

The term “Kabat numbering” unless otherwise stated, is defined as thenumbering of the residues in, e.g., an IgG heavy chain antibody usingthe EU index as in Kabat et al. (Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)), expressly incorporated herein by reference.

The term “Fc receptor” or “FcR” refers to a receptor that binds to theFc region of an antibody. Typical Fc receptors which bind to an Fcregion of an antibody (e.g., an IgG antibody) include, but are notlimited to, receptors of the FcγRI, FcγRII, and FcγRIII subclasses,including allelic variants and alternatively spliced forms of thesereceptors. Fc receptors are reviewed in Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); andde Haas et al., J. Lab. Clin. Med. 126:330-41 (1995).

I. Immunological and Therapeutic Reagents

Immunological and therapeutic reagents of the invention comprise orconsist of immunogens or antibodies, or functional or antigen bindingfragments thereof, as defined herein. The basic antibody structural unitis known to comprise a tetramer of subunits. Each tetramer is composedof two identical pairs of polypeptide chains, each pair having one“light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function.

Light chains are classified as either kappa or lambda and are about 230residues in length. Heavy chains are classified as gamma (γ), mu (μ),alpha (α), delta (δ), or epsilon (ε), are about 450-600 residues inlength, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE,respectively. Both heavy and light chains are folded into domains. Theterm “domain” refers to a globular region of a protein, for example, animmunoglobulin or antibody. Immunoglobulin or antibody domains include,for example, 3 or four peptide loops stabilized by β-pleated sheet andan interchain disulfide bond. Intact light chains have, for example, twodomains (V_(L) and C_(L)) and intact heavy chains have, for example,four or five domains (V_(H), C_(H)1, C_(H)2, and C_(H)3).

Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 10 more amino acids. (Seegenerally, Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press,N.Y. (1989), Ch. 7, incorporated by reference in its entirety for allpurposes).

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame. The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs.Naturally-occurring chains or recombinantly produced chains can beexpressed with a leader sequence which is removed during cellularprocessing to produce a mature chain. Mature chains can also berecombinantly produced having a non-naturally occurring leader sequence,for example, to enhance secretion or alter the processing of aparticular chain of interest.

The CDRs of the two mature chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. “FR4” also is referredto in the art as the D/J region of the variable heavy chain and the Jregion of the variable light chain. The assignment of amino acids toeach domain is in accordance with the definitions of Kabat, Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md., 1987 and 1991). An alternative structural definition hasbeen proposed by Chothia et al., J. Mol. Biol. 196:901 (1987); Nature342:878 (1989); and J. Mol. Biol. 186:651 (1989) (hereinaftercollectively referred to as “Chothia et al.” and incorporated byreference in their entirety for all purposes).

A. Aβ Antibodies

Therapeutic agents of the invention include antibodies that specificallybind to Aβ or other component of amyloid plaques. Such antibodies can bemonoclonal or polyclonal. Some such antibodies bind specifically to theaggregated form of Aβ without binding to the soluble form. Some bindspecifically to the soluble form without binding to the aggregated form.Some bind to both aggregated and soluble forms. Some such antibodiesbind to a naturally occurring short form of Aβ (i.e., Aβ39, 40 or 41)without binding to a naturally occurring long form of Aβ (i.e., Aβ42 andAβ43). Some antibodies bind to a long form of Aβ without binding to ashort form. Some antibodies bind to Aβ without binding to full-lengthamyloid precursor protein. Antibodies used in therapeutic methodspreferably have an intact constant region or at least sufficient of theconstant region to interact with an Fc receptor. Human isotype IgG1 ispreferred because of it having highest affinity of human isotypes forthe FcRI receptor on phagocytic cells. Bispecific Fab fragments can alsobe used, in which one arm of the antibody has specificity for Aβ, andthe other for an Fc receptor. Preferred antibodies bind to Aβ with abinding affinity greater than (or equal to) about 10⁶, 10⁷, 10⁸, 10⁹, or10¹⁰ M⁻¹ (including affinities intermediate of these values).

Polyclonal sera typically contain mixed populations of antibodiesbinding to several epitopes along the length of Aβ. However, polyclonalsera can be specific to a particular segment of Aβ, such as Aβ1-10.Monoclonal antibodies bind to a specific epitope within Aβ that can be aconformational or nonconformational epitope. Prophylactic andtherapeutic efficacy of antibodies can be tested using the transgenicanimal model procedures described in the Examples. Preferred monoclonalantibodies bind to an epitope within residues 1-10 of Aβ (with the firstN terminal residue of natural Aβ designated 1). Some preferredmonoclonal antibodies bind to an epitope within amino acids 1-5, andsome to an epitope within 5-10. Some preferred antibodies bind toepitopes within amino acids 1-3,1-4, 1-5,1-6, 1-7 or 3-7. Some preferredantibodies bind to an epitope starting at resides 1-3 and ending atresidues 7-11 of Aβ. Less preferred antibodies include those binding toepitopes with residues 10-15, 15-20, 25-30, 10-20, 20, 30, or 10-25 ofAβ. It is recommended that such antibodies be screened for activity inthe mouse models described in the Examples before use. For example, ithas been found that certain antibodies to epitopes within residues10-18, 16-24, 18-21 and 33-42 lack activity (e.g., lack the ability toreduce plaque burden and/or resolve the neuritic pathology associatedwith Alzheimer's disease). In some methods, multiple monoclonalantibodies having binding specificities to different epitopes are used.Such antibodies can be administered sequentially or simultaneously.Antibodies to amyloid components other than Aβ can also be used (e.g.,administered or co-administered). For example, antibodies can bedirected to the amyloid associated protein synuclein.

When an antibody is said to bind to an epitope within specifiedresidues, such as Aβ1-5 for example, what is meant is that the antibodyspecifically binds to a polypeptide containing the specified residues(i.e., Aβ1-5 in this an example). Such an antibody does not necessarilycontact every residue within Aβ1-5. Nor does every single amino acidsubstitution or deletion with in Aβ1-5 necessarily significantly affectbinding affinity. Epitope specificity of an antibody can be determined,for example, by forming a phage display library in which differentmembers display different subsequences of Aβ. The phage display libraryis then selected for members specifically binding to an antibody undertest. A family of sequences is isolated. Typically, such a familycontains a common core sequence, and varying lengths of flankingsequences in different members. The shortest core sequence showingspecific binding to the antibody defines the epitope bound by theantibody. Antibodies can also be tested for epitope specificity in acompetition assay with an antibody whose epitope specificity has alreadybeen determined. For example, antibodies that compete with the 3D6antibody for binding to Aβ bind to the same or similar epitope as 3D6,i.e., within residues Aβ1-5. Likewise antibodies that compete with the10D5 antibody bind to the same or similar epitope, i.e., within residuesAβ 3-7. Screening antibodies for epitope specificity is a usefulpredictor of therapeutic efficacy. For example, an antibody determinedto bind to an epitope within residues 1-7 of Aβ is likely to beeffective in preventing and treating Alzheimer's disease according tothe methodologies of the present invention.

Monoclonal or polyclonal antibodies that specifically bind to apreferred segment of Aβ without binding to other regions of Aβ have anumber of advantages relative to monoclonal antibodies binding to otherregions or polyclonal sera to intact Aβ. First, for equal mass dosages,dosages of antibodies that specifically bind to preferred segmentscontain a higher molar dosage of antibodies effective in clearingamyloid plaques. Second, antibodies specifically binding to preferredsegments can induce a clearing response against amyloid deposits withoutinducing a clearing response against intact APP polypeptide, therebyreducing the potential side effects.

1. Production of Nonhuman Antibodies

The present invention features non-human antibodies, for example,antibodies having specificity for the preferred Aβ epitopes of theinvention. Such antibodies can be used in formulating varioustherapeutic compositions of the invention or, preferably, providecomplementarity determining regions for the production of humanized orchimeric antibodies (described in detail below). The production ofnon-human monoclonal antibodies, e.g., murine, guinea pig, primate,rabbit or rat, can be accomplished by, for example, immunizing theanimal with Aβ. A longer polypeptide comprising Aβ or an immunogenicfragment of Aβ or anti-idiotypic antibodies to an antibody to Aβ canalso be used. See Harlow & Lane, supra, incorporated by reference forall purposes). Such an immunogen can be obtained from a natural source,by peptide synthesis or by recombinant expression. Optionally, theimmunogen can be administered fused or otherwise complexed with acarrier protein, as described below. Optionally, the immunogen can beadministered with an adjuvant. The term “adjuvant” refers to a compoundthat when administered in conjunction with an antigen augments theimmune response to the antigen, but when administered alone does notgenerate an immune response to the antigen. Adjuvants can augment animmune response by several mechanisms including lymphocyte recruitment,stimulation of B and/or T cells, and stimulation of macrophages. Severaltypes of adjuvant can be used as described below. Complete Freund'sadjuvant followed by incomplete adjuvant is preferred for immunizationof laboratory animals.

Rabbits or guinea pigs are typically used for making polyclonalantibodies. Exemplary preparation of polyclonal antibodies, e.g., forpassive protection, can be performed as follows. 125 non-transgenic miceare immunized with 100 μg Aβ1-42, plus CFA/IFA adjuvant, and euthanizedat 4-5 months. Blood is collected from immunized mice. IgG is separatedfrom other blood components. Antibody specific for the immunogen may bepartially purified by affinity chromatography. An average of about 0.5-1mg of immunogen-specific antibody is obtained per mouse, giving a totalof 60-120 mg.

Mice are typically used for making monoclonal antibodies. Monoclonalscan be prepared against a fragment by injecting the fragment or longerform of Aβ into a mouse, preparing hybridomas and screening thehybridomas for an antibody that specifically binds to Aβ. Optionally,antibodies are screened for binding to a specific region or desiredfragment of Aβ without binding to other nonoverlapping fragments of Aβ.The latter screening can be accomplished by determining binding of anantibody to a collection of deletion mutants of an Aβ peptide anddetermining which deletion mutants bind to the antibody. Binding can beassessed, for example, by Western blot or ELISA. The smallest fragmentto show specific binding to the antibody defines the epitope of theantibody. Alternatively, epitope specificity can be determined by acompetition assay is which a test and reference antibody compete forbinding to Aβ. If the test and reference antibodies compete, then theybind to the same epitope or epitopes sufficiently proximal such thatbinding of one antibody interferes with binding of the other. Thepreferred isotype for such antibodies is mouse isotype IgG2a orequivalent isotype in other species. Mouse isotype IgG2a is theequivalent of human isotype IgG1.

2. Chimeric and Humanized Antibodies

The present invention also features chimeric and/or humanized antibodies(i.e., chimeric and/or humanized immunoglobulins) specific for betaamyloid peptide. Chimeric and/or humanized antibodies have the same orsimilar binding specificity and affinity as a mouse or other nonhumanantibody that provides the starting material for construction of achimeric or humanized antibody.

A. Production of Chimeric Antibodies

The term “chimeric antibody” refers to an antibody whose light and heavychain genes have been constructed, typically by genetic engineering,from immunoglobulin gene segments belonging to different species. Forexample, the variable (V) segments of the genes from a mouse monoclonalantibody may be joined to human constant (C) segments, such as IgG1 andIgG4. Human isotype IgG1 is preferred. A typical chimeric antibody isthus a hybrid protein consisting of the V or antigen-binding domain froma mouse antibody and the C or effector domain from a human antibody.

B. Production of Humanized Antibodies

The term “humanized antibody” refers to an antibody comprising at leastone chain comprising variable region framework residues substantiallyfrom a human antibody chain (referred to as the acceptor immunoglobulinor antibody) and at least one complementarity determining regionsubstantially from a mouse-antibody, (referred to as the donorimmunoglobulin or antibody). See, Queen et al., Proc. Natl. Acad. Sci.USA 86:10029-10033 (1989), U.S. Pat. No. 5,530,101, U.S. Pat. No.5,585,089, U.S. Pat. No. 5,693,761, U.S. Pat. No. 5,693,762, Selick etal., WO 90/07861, and Winter, U.S. Pat. No. 5,225,539 (incorporated byreference in their entirety for all purposes). The constant region(s),if present, are also substantially or entirely from a humanimmunoglobulin.

The substitution of mouse CDRs into a human variable domain framework ismost likely to result in retention of their correct spatial orientationif the human variable domain framework adopts the same or similarconformation to the mouse variable framework from which the CDRsoriginated. This is achieved by obtaining the human variable domainsfrom human antibodies whose framework sequences exhibit a high degree ofsequence identity with the murine variable framework domains from whichthe CDRs were derived. The heavy and light chain variable frameworkregions can be derived from the same or different human antibodysequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies. See Kettleborough et al., Protein Engineering4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993) andCarter et al., WO 92/22653.

Having identified the complementarity determining regions of the murinedonor immunoglobulin and appropriate human acceptor immunoglobulins, thenext step is to determine which, if any, residues from these componentsshould be substituted to optimize the properties of the resultinghumanized antibody. In general, substitution of human amino acidresidues with murine should be minimized, because introduction of murineresidues increases the risk of the antibody eliciting ahuman-anti-mouse-antibody (HAMA) response in humans. Art-recognizedmethods of determining immune response can be performed to monitor aHAMA response in a particular patient or during clinical trials.Patients administered humanized antibodies can be given animmunogenicity assessment at the beginning and throughout theadministration of said therapy. The HAMA response is measured, forexample, by detecting antibodies to the humanized therapeutic reagent,in serum samples from the patient using a method known to one in theart, including surface plasmon resonance technology (BIACORE) and/orsolid-phase ELISA analysis.

Certain amino acids from the human variable region framework residuesare selected for substitution based on their possible influence on CDRconformation and/or binding to antigen. The unnatural juxtaposition ofmurine CDR regions with human variable framework region can result inunnatural conformational restraints, which, unless corrected bysubstitution of certain amino acid residues, lead to loss of bindingaffinity.

The selection of amino acid residues for substitution is determined, inpart, by computer modeling. Computer hardware and software are describedherein for producing three-dimensional images of immunoglobulinmolecules. In general, molecular models are produced starting fromsolved structures for immunoglobulin chains or domains thereof. Thechains to be modeled are compared for amino acid sequence similaritywith chains or domains of solved three-dimensional structures, and thechains or domains showing the greatest sequence similarity is/areselected as starting points for construction of the molecular model.Chains or domains sharing at least 50% sequence identity are selectedfor modeling, and preferably those sharing at least 60%, 70%, 80%, 90%sequence identity or more are selected for modeling. The solved startingstructures are modified to allow for differences between the actualamino acids in the immunoglobulin chains or domains being modeled, andthose in the starting structure. The modified structures are thenassembled into a composite immunoglobulin. Finally, the model is refinedby energy minimization and by verifying that all atoms are withinappropriate distances from one another and that bond lengths and anglesare within chemically acceptable limits.

The selection of amino acid residues for substitution can also bedetermined, in part, by examination of the characteristics of the aminoacids at particular locations, or empirical observation of the effectsof substitution or mutagenesis of particular amino acids. For example,when an amino acid differs between a murine variable region frameworkresidue and a selected human variable region framework residue, thehuman framework amino acid should usually be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

(1) noncovalently binds antigen directly,

(2) is adjacent to a CDR region,

(3) otherwise interacts with a CDR region (e.g., is within about 3-6 Åof a CDR region as determined by computer modeling), or

(4) participates in the VL-VH interface.

Residues which “noncovalently bind antigen directly” include amino acidsin positions in framework regions which are have a good probability ofdirectly interacting with amino acids on the antigen according toestablished chemical forces, for example, by hydrogen bonding, Van derWaals forces, hydrophobic interactions, and the like.

CDR and framework regions are as defined by Kabat et al. or Chothia etal., supra. When framework residues, as defined by Kabat et al., supra,constitute structural loop residues as defined by Chothia et al., supra,the amino acids present in the mouse antibody may be selected forsubstitution into the humanized antibody. Residues which are “adjacentto a CDR region” include amino acid residues in positions immediatelyadjacent to one or more of the CDRs in the primary sequence of thehumanized immunoglobulin chain, for example, in positions immediatelyadjacent to a CDR as defined by Kabat, or a CDR as defined by Chothia(See e.g., Chothia and Lesk JMB 196:901 (1987)). These amino acids areparticularly likely to interact with the amino acids in the CDRs and, ifchosen from the acceptor, to distort the donor CDRs and reduce affinity.Moreover, the adjacent amino acids may interact directly with theantigen (Amit et al., Science, 233:747 (1986), which is incorporatedherein by reference) and selecting these amino acids from the donor maybe desirable to keep all the antigen contacts that provide affinity inthe original antibody.

Residues that “otherwise interact with a CDR region” include those thatare determined by secondary structural analysis to be in a spatialorientation sufficient to effect a CDR region. In one embodiment,residues that “otherwise interact with a CDR region” are identified byanalyzing a three-dimensional model of the donor immunoglobulin (e.g., acomputer-generated model). A three-dimensional model, typically of theoriginal donor antibody, shows that certain amino acids outside of theCDRs are close to the CDRs and have a good probability of interactingwith amino acids in the CDRs by hydrogen bonding, Van der Waals forces,hydrophobic interactions, etc. At those amino acid positions, the donorimmunoglobulin amino acid rather than the acceptor immunoglobulin aminoacid may be selected. Amino acids according to this criterion willgenerally have a side chain atom within about 3 angstrom units (A) ofsome atom in the CDRs and must contain an atom that could interact withthe CDR atoms according to established chemical forces, such as thoselisted above.

In the case of atoms that may form a hydrogen bond, the 3 Å is measuredbetween their nuclei, but for atoms that do not form a bond, the 3 Å ismeasured between their Van der Waals surfaces. Hence, in the lattercase, the nuclei must be within about 6 Å (3 Å plus the sum of the Vander Waals radii) for the atoms to be considered capable of interacting.In many cases the nuclei will be from 4 or 5 to 6 Å apart. Indetermining whether an amino acid can interact with the CDRs, it ispreferred not to consider the last 8 amino acids of heavy chain CDR 2 aspart of the CDRs, because from the viewpoint of structure, these 8 aminoacids behave more as part of the framework.

Amino acids that are capable of interacting with amino acids in theCDRs, may be identified in yet another way. The solvent accessiblesurface area of each framework amino acid is calculated in two ways: (1)in the intact antibody, and (2) in a hypothetical molecule consisting ofthe antibody with its CDRs removed. A significant difference betweenthese numbers of about 10 square angstroms or more shows that access ofthe framework amino acid to solvent is at least partly blocked by theCDRs, and therefore that the amino acid is making contact with the CDRs.Solvent accessible surface area of an amino acid may be calculated basedon a three-dimensional model of an antibody, using algorithms known inthe art (e.g., Connolly, J. Appl. Cryst. 16:548 (1983) and Lee andRichards, J. Mol. Biol. 55:379 (1971), both of which are incorporatedherein by reference). Framework amino acids may also occasionallyinteract with the CDRs indirectly, by affecting the conformation ofanother framework amino acid that in turn contacts the CDRs.

The amino acids at several positions in the framework are known to becapable of interacting with the CDRs in many antibodies (Chothia andLesk, supra, Chothia et al., supra and Tramontano et al., J. Mol. Biol.215:175 (1990), all of which are incorporated herein by reference).Notably, the amino acids at positions 2, 48, 64 and 71 of the lightchain and 26-30, 71 and 94 of the heavy chain (numbering according toKabat) are known to be capable of interacting with the CDRs in manyantibodies. The amino acids at positions 35 in the light chain and 93and 103 in the heavy chain are also likely to interact with the CDRs. Atall these numbered positions, choice of the donor amino acid rather thanthe acceptor amino acid (when they differ) to be in the humanizedimmunoglobulin is preferred. On the other hand, certain residues capableof interacting with the CDR region, such as the first 5 amino acids ofthe light chain, may sometimes be chosen from the acceptorimmunoglobulin without loss of affinity in the humanized immunoglobulin.

Residues which “participate in the VL−VH interface” or “packingresidues” include those residues at the interface between VL and VH asdefined, for example, by Novotny and Haber, Proc. Natl. Acad. Sci. USA,82:4592-66 (1985) or Chothia et al, supra. Generally, unusual packingresidues should be retained in the humanized antibody if they differfrom those in the human frameworks.

In general, one or more of the amino acids fulfilling the above criteriais substituted. In some embodiments, all or most of the amino acidsfulfilling the above criteria are substituted. Occasionally, there issome ambiguity about whether a particular amino acid meets the abovecriteria, and alternative variant immunoglobulins are produced, one ofwhich has that particular substitution, the other of which does not.Alternative variant immunoglobulins so produced can be tested in any ofthe assays described herein for the desired activity, and the preferredimmunoglobulin selected.

Usually the CDR regions in humanized antibodies are substantiallyidentical, and more usually, identical to the corresponding CDR regionsof the donor antibody. Although not usually desirable, it is sometimespossible to make one or more conservative amino acid substitutions ofCDR residues without appreciably affecting the binding affinity of theresulting humanized immunoglobulin. By conservative substitutions isintended combinations such as gly, ala; val, ile, leu; asp, glu; asn,gln; ser, thr; lys, arg; and phe, tyr.

Additional candidates for substitution are acceptor human frameworkamino acids that are unusual or “rare” for a human immunoglobulin atthat position. These amino acids can be substituted with amino acidsfrom the equivalent position of the mouse donor antibody or from theequivalent positions of more typical human immunoglobulins. For example,substitution may be desirable when the amino acid in a human frameworkregion of the acceptor immunoglobulin is rare for that position and thecorresponding amino acid in the donor immunoglobulin is common for thatposition in human immunoglobulin sequences; or when the amino acid inthe acceptor immunoglobulin is rare for that position and thecorresponding amino acid in the donor immunoglobulin is also rare,relative to other human sequences. These criterion help ensure that anatypical amino acid in the human framework does not disrupt the antibodystructure. Moreover, by replacing an unusual human acceptor amino acidwith an amino acid from the donor antibody that happens to be typicalfor human antibodies, the humanized antibody may be made lessimmunogenic.

The term “rare”, as used herein, indicates an amino acid occurring atthat position in less than about 20% but usually less than about 10% ofsequences in a representative sample of sequences, and the term“common”, as used herein, indicates an amino acid occurring in more thanabout 25% but usually more than about 50% of sequences in arepresentative sample. For example, all human light and heavy chainvariable region sequences are respectively grouped into “subgroups” ofsequences that are especially homologous to each other and have the sameamino acids at certain critical positions (Kabat et al., supra). Whendeciding whether an amino acid in a human acceptor sequence is “rare” or“common” among human sequences, it will often be preferable to consideronly those human sequences in the same subgroup as the acceptorsequence.

Additional candidates for substitution are acceptor human frameworkamino acids that would be identified as part of a CDR region under thealternative definition proposed by Chothia et al., supra. Additionalcandidates for substitution are acceptor human framework amino acidsthat would be identified as part of a CDR region under the AbM and/orcontact definitions. Notably, CDR1 in the variable heavy chain isdefined as including residues 26-32.

Additional candidates for substitution are acceptor framework residuesthat correspond to a rare or unusual donor framework residue. Rare orunusual donor framework residues are those that are rare or unusual (asdefined herein) for murine antibodies at that position. For murineantibodies, the subgroup can be determined according to Kabat andresidue positions identified which differ from the consensus. Thesedonor specific differences may point to somatic mutations in the murinesequence which enhance activity. Unusual residues that are predicted toaffect binding are retained, whereas residues predicted to beunimportant for binding can be substituted.

Additional candidates for substitution are non-germline residuesoccurring in an acceptor framework region. For example, when an acceptorantibody chain (i.e., a human antibody chain sharing significantsequence identity with the donor antibody chain) is aligned to agermline antibody chain (likewise sharing significant sequence identitywith the donor chain), residues not matching between acceptor chainframework and the germline chain framework can be substituted withcorresponding residues from the germline sequence.

Other than the specific amino acid substitutions discussed above, theframework regions of humanized immunoglobulins are usually substantiallyidentical, and more usually, identical to the framework regions of thehuman antibodies from which they were derived. Of course, many of theamino acids in the framework region make little or no directcontribution to the specificity or affinity of an antibody. Thus, manyindividual conservative substitutions of framework residues can betolerated without appreciable change of the specificity or affinity ofthe resulting humanized immunoglobulin. Thus, in one embodiment thevariable framework region of the humanized immunoglobulin shares atleast 85% sequence identity to a human variable framework regionsequence or consensus of such sequences. In another embodiment, thevariable framework region of the humanized immunoglobulin shares atleast 90%, preferably 95%, more preferably 96%, 97%, 98% or 99% sequenceidentity to a human variable framework region sequence or consensus ofsuch sequences. In general, however, such substitutions are undesirable.

The humanized antibodies preferably exhibit a specific binding affinityfor antigen of at least 10⁷, 10⁸, 10⁹ or 10¹⁰ M⁻¹. Usually the upperlimit of binding affinity of the humanized antibodies for antigen iswithin a factor of three, four or five of that of the donorimmunoglobulin. Often the lower limit of binding affinity is also withina factor of three, four or five of that of donor immunoglobulin.Alternatively, the binding affinity can be compared to that of ahumanized antibody having no substitutions (e.g., an antibody havingdonor CDRs and acceptor FRs, but no FR substitutions). In suchinstances, the binding of the optimized antibody (with substitutions) ispreferably at least two- to three-fold greater, or three- to four-foldgreater, than that of the unsubstituted antibody. For makingcomparisons, activity of the various antibodies can be determined, forexample, by BIACORE (i.e., surface plasmon resonance using unlabelledreagents) or competitive binding assays.

C. Production of Humanized 3D6 Antibodies

A preferred embodiment of the present invention features a humanizedantibody to the N-terminus of Aβ, in particular, for use in thetherapeutic and/or diagnostic methodologies described herein. Aparticularly preferred starting material for production of humanizedantibodies is 3D6. 3D6 is specific for the N-terminus of Aβ and has beenshown to mediate phagocytosis (e.g., induce phagocytosis) of amyloidplaque (see Examples I-V). The cloning and sequencing of cDNA encodingthe 3D6 antibody heavy and light chain variable regions is described inExample VI.

Suitable human acceptor antibody sequences are identified by computercomparisons of the amino acid sequences of the mouse variable regionswith the sequences of known human antibodies. The comparison isperformed separately for heavy and light chains but the principles aresimilar for each. In particular, variable domains from human antibodieswhose framework sequences exhibit a high degree of sequence identitywith the murine VL and VH framework regions were identified by query ofthe Kabat Database using NCBI BLAST (publicly accessible through theNational Institutes of Health NCBI internet server) with the respectivemurine framework sequences. In one embodiment, acceptor sequencessharing greater that 50% sequence identity with murine donor sequencesare selected. Preferably, acceptor antibody sequences sharing 60%, 70%,80%, 90% or more are selected.

A computer comparison of 3D6 revealed that the 3D6 light chain shows thegreatest sequence identity to human light chains of subtype kappa II,and that the 3D6 heavy chain shows greatest sequence identity to humanheavy chains of subtype III, as defined by Kabat et al., supra. Thus,light and heavy human framework regions are preferably derived fromhuman antibodies of these subtypes, or from consensus sequences of suchsubtypes. The preferred light chain human variable regions showinggreatest sequence identity to the corresponding region from 3D6 are fromantibodies having Kabat ID Numbers 019230, 005131, 005058, 005057,005059, U21040 and U41645, with 019230 being more preferred. Thepreferred heavy chain human variable regions showing greatest sequenceidentity to the corresponding region from 3D6 are from antibodies havingKabat ID Numbers 045919, 000459, 000553, 000386 and M23691, with 045919being more preferred.

Residues are next selected for substitution, as follows. When an aminoacid differs between a 3D6 variable framework region and an equivalenthuman variable framework region, the human framework amino acid shouldusually be substituted by the equivalent mouse amino acid if it isreasonably expected that the amino acid:

(1) noncovalently binds antigen directly,

(2) is adjacent to a CDR region, is part of a CDR region under thealternative definition proposed by Chothia et al., supra, or otherwiseinteracts with a CDR region (e.g., is within about 3A of a CDR region)(e.g., amino acids at positions L2, H49 and H94 of 3D6), or

(3) participates in the VL−VH interface (e.g., amino acids at positionsL36, L46 and H93 of 3D6).

Computer modeling of the 3D6 antibody heavy and light chain variableregions, and humanization of the 3D6 antibody is described in ExampleVII. Briefly, a three-dimensional model was generated based on theclosest solved murine antibody structures for the heavy and lightchains. For this purpose, an antibody designated 1CR9 (Protein Data Bank(PDB) ID: 1CR9, Kanyo et al., J. Mol. Biol. 293:855 (1999)) was chosenas a template for modeling the 3D6 light chain, and an antibodydesignated 1OPG (PDB ID: 1OPG, Kodandapani et al., J. Biol. Chem.270:2268 (1995)) was chosen as the template for modeling the heavychain. The model was further refined by a series of energy minimizationsteps to relieve unfavorable atomic contacts and optimize electrostaticand van der Walls interactions. The solved structure of 1qkz (PDB ID:1QKZ, Derrick et al., J. Mol. Biol. 293:81 (1999)) was chosen as atemplate for modeling CDR3 of the heavy chain as 3D6 and 1OPG did notexhibit significant sequence homology in this region when aligned forcomparison purposes.

Three-dimensional structural information for the antibodies describedherein is publicly available, for example, from the ResearchCollaboratory for Structural Bioinformatics' Protein Data Bank (PDB).The PDB is freely accessible via the World Wide Web internet and isdescribed by Berman et al. (2000) Nucleic Acids Research, 28:235.Computer modeling allows for the identification of CDR-interactingresidues. The computer model of the structure of 3D6 can in turn serveas a starting point for predicting the three-dimensional structure of anantibody containing the 3D6 complementarity determining regionssubstituted in human framework structures. Additional models can beconstructed representing the structure as further amino acidsubstitutions are introduced.

In general, substitution of one, most or all of the amino acidsfulfilling the above criteria is desirable. Accordingly, the humanizedantibodies of the present invention will usually contain a substitutionof a human light chain framework residue with a corresponding 3D6residue in at least 1, 2 or 3, and more usually 4, of the followingpositions: L1, L2, L36 and L46. The humanized antibodies also usuallycontain a substitution of a human heavy chain framework residue with acorresponding 3D6 residue in at least 1, 2, and sometimes 3, of thefollowing positions: H49, H93 and H94. Humanized antibodies can alsocontain a substitution of a heavy chain framework residue with acorresponding germline residue in at least 1, 2, and sometimes 3, of thefollowing positions: H74, H77 and H89.

Occasionally, however, there is some ambiguity about whether aparticular amino acid meets the above criteria, and alternative variantimmunoglobulins are produced, one of which has that particularsubstitution, the other of which does not. In instances wheresubstitution with a murine residue would introduce a residue that israre in human immunoglobulins at a particular position, it may bedesirable to test the antibody for activity with or without theparticular substitution. If activity (e.g., binding affinity and/orbinding specificity) is about the same with or without the substitution,the antibody without substitution may be preferred, as it would beexpected to elicit less of a HAHA response, as described herein.

Other candidates for substitution are acceptor human framework aminoacids that are unusual for a human immunoglobulin at that position.These amino acids can be substituted with amino acids from theequivalent position of more typical human immunoglobulins.Alternatively, amino acids from equivalent positions in the mouse 3D6can be introduced into the human framework regions when such amino acidsare typical of human immunoglobulin at the equivalent positions.

In additional embodiments, when the human light chain framework acceptorimmunoglobulin is Kabat ID Number 019230, the light chain containssubstitutions in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or moreusually 13, of the following positions: L7, L10, L12, L15, L17, L39,L45, L63, L78, L83, L85, L100 or L104. In additional embodiments whenthe human heavy chain framework acceptor immunoglobulin is Kabat IDNumber 045919, the heavy chain contains substitutions in at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more usually 15, of thefollowing positions: H3, H5, H13, H16, H19, H40, H41, H42, H44, H72,H77, H82A, H83, H84, or H108. These positions are substituted with theamino acid from the equivalent position of a human immunoglobulin havinga more typical amino acid residue. Examples of appropriate amino acidsto substitute are shown in FIGS. 1 and 2.

Other candidates for substitution are non-germline residues occurring ina framework region. A computer comparison of 3D6 with known germlinesequences revealed that heavy chains showing the greatest degree ofsequence identity include germline variable region sequences VH3-48,VH3-23, VH3-7, VH3-21 and VH3-11, with VH3-23 being more preferred.Alignment of Kabat ID 045919 with VH3-23 reveals that residues H74, H77and/or H89 may be selected for substitution with corresponding germlineresidues (e.g., residues H74, H77 and/or H89 when comparing Kabat ID045919 and VH3-23). Likewise, germline sequences having the greatestdegree of identity to the 3D6 light chain include A1, A17, A18, A2 andA19, with A19 being most preferred. Residues not matching between aselected light chain acceptor framework and one of these germlinesequences could be selected for substitution with the correspondinggermline residue.

Table 1 summarizes the sequence analysis of the 3D6 VH and VL regions.Additional mouse and human structures that can be used for computermodeling of the 3D6 antibody and additional human antibodies are setforth as well as germline sequences that can be used in selecting aminoacid substitutions. Rare mouse residues are also set forth in Table 1.Rare mouse residues are identified by comparing the donor VL and/or VHsequences with the sequences of other members of the subgroup to whichthe donor VL and/or VH sequences belong (according to Kabat) andidentifying the residue positions which differ from the consensus. Thesedonor specific differences may point to somatic mutations which enhanceactivity. Unusual or rare residues close to the binding site maypossibly contact the antigen, making it desirable to retain the mouseresidue. However, if the unusual mouse residue is not important forbinding, use of the corresponding acceptor residue is preferred as themouse residue may create immunogenic neoepitopes in the humanizedantibody. In the situation where an unusual residue in the donorsequence is actually a common residues in the corresponding acceptorsequence, the preferred residue is clearly the acceptor residue. TABLE 1Summary of 3D6 V-region sequence Chain Heavy Light Mouse subgroup IIID(002688) II (005840-005844, 005851-005853, (Kabat seq ID#) 005857,005863) Mouse homologs 002727/163.1′CL 005840/1210.7 (Kabat/Genbank)002711/H35-C6′CL 005843/42.4b.12.2′CL 002733/8-1-12-5-3-1(A2-1)′CL005842/BXW-14′CL 002715/ASWA2′CL 005841/42.7B3.2′CL 020669/#14′CL005851/36-60CRI- Rare amino acids (% N40 (0.233%) Y1(.035%) frequency ofD42 (0.699%) I15 (3.3%) occurrence in class) D27 (0.867%)-CDR1 I78(0.677%) L85 (0.625%) W89 (0.815%)-CDR3 K106A (0.295%) Human SubgroupIII (000488-000491, 000503, 000624) II (005046) Chothia canonical H1:class 1 [2fbj] L1: class 4 [1rmf] CDR groupings [pdb H2: class 3 [1igc]L2: class1 [1lmk] example] L3: class 1 [1tet] Closest solved mouse PDBID: 1OPG Kodandapani et al., PDB ID: 1CR9; Kanyo et al., supra;structures supra; (72% 2 Å) (94%, 2 Å) PDB ID: 1NLD; Davies et al., ActaCrystallogr. D. Biol. Crystallog. 53: 186 (1997); (98%, 2.8 Å) Closestsolved human 1VH (68%, nmr) 1LVE (57%, LEN) structures 443560 (65%, IgG,λ myeloma, 1.8 Å) 1B6DA (54%, B-J dimer, 2.8 Å); KOL/2FB4H (60%,myeloma, 3 Å) 1VGEL (54%, autoAb) Germline query (Hu) VH3-48(4512283/BAA75032.1) A1(x63402) results (top 4) VH3-23(4512287/BAA75046.1) A17 (x63403) VH3-7 (4512300/BAA75056.1) A18(X63396) VH3-21 (4512287/BAA75047.1) A2 (m31952) VH3-11(4152300/BAA75053.1) A19 (x63397)*heavy chain and light chain from the same antibody (O-81, Hirabayashiet al. NAR 20: 2601).

Kabat ID sequences referenced herein are publicly available, forexample, from the Northwestern University Biomedical EngineeringDepartment's Kabat Database of Sequences of Proteins of ImmunologicalInterest. Three-dimensional structural information for antibodiesdescribed herein is publicly available, for example, from the ResearchCollaboratory for Structural Bioinformatics' Protein Data Bank (PDB).The PDB is freely accessible via the World Wide Web internet and isdescribed by Berman et al. (2000) Nucleic Acids Research, p 235-242.Germline gene sequences referenced herein are publicly available, forexample, from the National Center for Biotechnology Information (NCBI)database of sequences in collections of Igh, Ig kappa and Ig lambdagermline V genes (as a division of the National Library of Medicine(NLM) at the National Institutes of Health (NIH)). Homology searching ofthe NCBI “Ig Germline Genes” database is provided by IgG BLAST™.

In a preferred embodiment, a humanized antibody of the present inventioncontains (i) a light chain comprising a variable domain comprisingmurine 3D6 VL CDRs and a human acceptor framework, the framework havingat least one, preferably two, three or four residues selected from thegroup consisting of L1, L2, L36, and L46 substituted with thecorresponding 3D6 residue and (ii) a heavy chain comprising 3D6 VH CDRsand a human acceptor framework, the framework having at least one,preferably two or three residues selected from the group consisting ofH49, H93 and H94 substituted with the corresponding 3D6 residue, and,optionally, at least one, preferably two or three residues selected fromthe group consisting of H74, H77 and H89 is substituted with acorresponding human germline residue.

In a more preferred embodiment, a humanized antibody of the presentinvention contains (i) a light chain comprising a variable domaincomprising murine 3D6 VL CDRs and a human acceptor framework, theframework having residue 1 substituted with a tyr (Y), residue 2substituted with a val (V), residue 36 substituted with a leu (L) and/orresidue 46 substituted with an arg (R), and (ii) a heavy chaincomprising 3D6 VH CDRs and a human acceptor framework, the frameworkhaving residue 49 substituted with an ala (A), residue 93 substitutedwith a val (V) and/or residue 94 substituted with an arg (R), and,optionally, having residue 74 substituted with a ser (S), residue 77substituted with a thr (T) and/or residue 89 substituted with a val (V).

In a particularly preferred embodiment, a humanized antibody of thepresent invention has structural features, as described herein, andfurther has at least one (preferably two, three, four or all) of thefollowing activities: (1) binds aggregated Aβ1-42 (e.g., as determinedby ELISA); (2) binds Aβ in plaques (e.g., staining of AD and/or PDAPPplaques); (3) binds Aβ with two- to three- fold higher binding affinityas compared to chimeric 3D6 (e.g., 3D6 having murine variable regionsequences and human constant region sequences); (4) mediatesphagocytosis of Aβ (e.g., in an ex vivo phagocytosis assay, as describedherein); and (5) crosses the blood-brain barrier (e.g., demonstratesshort-term brain localization, for example, in a PDAPP animal model, asdescribed herein).

In another embodiment, a humanized antibody of the present invention hasstructural features, as described herein, binds Aβ in a manner or withan affinity sufficient to elicit at least one of the following in vivoeffects: (1) reduce Aβ plaque burden; (2) prevent plaque formation; (3)reduce levels of soluble Aβ; (4) reduce the neuritic pathologyassociated with an amyloidogenic disorder; (5) lessens or ameliorate atleast one physiological symptom associated with an amyloidogenicdisorder; and/or (6) improves cognitive function.

In another embodiment, a humanized antibody of the present invention hasstructural features, as described herein, and specifically binds to anepitope comprising residues 1-5 or 3-7 of Aβ.

The activities described above can be determined utilizing any one of avariety of assays described herein or in the art (e.g., binding assays,phagocytosis assays, etc.). Activities can be assayed either in vivo(e.g., using labeled assay components and/or imaging techniques) or invitro (e.g., using samples or specimens derived from a subject).Activities can be assayed either directly or indirectly. In certainpreferred embodiments, neurological endpoints (e.g., amyloid burden,neuritic burden, etc) are assayed. Such endpoints can be assayed inliving subjects (e.g., in animal models of Alzheimer's disease or inhuman subjects, for example, undergoing immunotherapy) usingnon-invasive detection methodologies. Alternatively, such endpoints canbe assayed in subjects post mortem. Assaying such endpoints in animalmodels and/or in human subjects post mortem is useful in assessing theeffectiveness of various agents (e.g., humanized antibodies) to beutilized in similar immunotherapeutic applications. In other preferredembodiments, behavioral or neurological parameters can be assessed asindicators of the above neuropathological activities or endpoints.

3. Human Antibodies

Human antibodies against Aβ are provided by a variety of techniquesdescribed below. Some human antibodies are selected by competitivebinding experiments, or otherwise, to have the same epitope specificityas a particular mouse antibody, such as one of the mouse monoclonalsdescribed herein. Human antibodies can also be screened for a particularepitope specificity by using only a fragment of Aβ as the immunogen,and/or by screening antibodies against a collection of deletion mutantsof Aβ. Human antibodies preferably have human IgG1 isotype specificity.

a. Trioma Methodology

The basic approach and an exemplary cell fusion partner, SPAZ-4, for usein this approach have been described by Oestberg et al., Hybridoma 2:361(1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman et al., U.S.Pat. No. 4,634,666 (each of which is incorporated by reference in itsentirety for all purposes). The antibody-producing cell lines obtainedby this method are called triomas, because they are descended from threecells; two human and one mouse. Initially, a mouse myeloma line is fusedwith a human B-lymphocyte to obtain a non-antibody-producing xenogeneichybrid cell, such as the SPAZ-4 cell line described by Oestberg, supra.The xenogeneic cell is then fused with an immunized human B-lymphocyteto obtain an antibody-producing trioma cell line. Triomas have beenfound to produce antibody more stably than ordinary hybridomas made fromhuman cells.

The immunized B-lymphocytes are obtained from the blood, spleen, lymphnodes or bone marrow of a human donor. If antibodies against a specificantigen or epitope are desired, it is preferable to use that antigen orepitope thereof for immunization. Immunization can be either in vivo orin vitro. For in vivo immunization, B cells are typically isolated froma human immunized with Aβ, a fragment thereof, larger polypeptidecontaining Aβ or fragment, or an anti-idiotypic antibody to an antibodyto Aβ. In some methods, B cells are isolated from the same patient whois ultimately to be administered antibody therapy. For in vitroimmunization, B-lymphocytes are typically exposed to antigen for aperiod of 7-14 days in a media such as RPMI-1640 (see Engleman, supra)supplemented with 10% human plasma.

The immunized B-lymphocytes are fused to a xenogeneic hybrid cell suchas SPAZ-4 by well-known methods. For example, the cells are treated with40-50% polyethylene glycol of MW 1000-4000, at about 37 degrees C., forabout 5-10 min. Cells are separated from the fusion mixture andpropagated in media selective for the desired hybrids (e.g., HAT or AH).Clones secreting antibodies having the required binding specificity areidentified by assaying the trioma culture medium for the ability to bindto Aβ or a fragment thereof. Triomas producing human antibodies havingthe desired specificity are subcloned by the limiting dilution techniqueand grown in vitro in culture medium. The trioma cell lines obtained arethen tested for the ability to bind Aβ or a fragment thereof.

Although triomas are genetically stable they do not produce antibodiesat very high levels. Expression levels can be increased by cloningantibody genes from the trioma into one or more expression vectors, andtransforming the vector into standard mammalian, bacterial or yeast celllines.

b. Transgenic Non-Human Mammals

Human antibodies against Aβ can also be produced from non-humantransgenic mammals having transgenes encoding at least a segment of thehuman immunoglobulin locus. Usually, the endogenous immunoglobulin locusof such transgenic mammals is functionally inactivated. Preferably, thesegment of the human immunoglobulin locus includes unrearrangedsequences of heavy and light chain components. Both inactivation ofendogenous immunoglobulin genes and introduction of exogenousimmunoglobulin genes can be achieved by targeted homologousrecombination, or by introduction of YAC chromosomes. The transgenicmammals resulting from this process are capable of functionallyrearranging the immunoglobulin component sequences, and expressing arepertoire of antibodies of various isotypes encoded by humanimmunoglobulin genes, without expressing endogenous immunoglobulingenes. The production and properties of mammals having these propertiesare described in detail by, e.g., Lonberg et al., WO93/12227 (1993);U.S. Pat. No. 5,877,397, U.S. Pat. No. 5,874,299, U.S. Pat. No.5,814,318, U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,770,429, U.S. Pat.No. 5,661,016, U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,625,126, U.S.Pat. No. 5,569,825, U.S. Pat. No. 5,545,806, Nature 148:1547 (1994),Nature Biotechnology 14:826 (1996), Kucherlapati, WO 91/10741 (1991)(each of which is incorporated by reference in its entirety for allpurposes). Transgenic mice are particularly suitable. Anti-Aβ antibodiesare obtained by immunizing a transgenic nonhuman mammal, such asdescribed by Lonberg or Kucherlapati, supra, with Aβ or a fragmentthereof. Monoclonal antibodies are prepared by, e.g., fusing B-cellsfrom such mammals to suitable myeloma cell lines using conventionalKohler-Milstein technology. Human polyclonal antibodies can also beprovided in the form of serum from humans immunized with an immunogenicagent. Optionally, such polyclonal antibodies can be concentrated byaffinity purification using Aβ or other amyloid peptide as an affinityreagent.

c. Phage Display Methods

A further approach for obtaining human anti-Aβ antibodies is to screen aDNA library from human B cells according to the general protocoloutlined by Huse et al., Science 246:1275-1281 (1989). As described fortrioma methodology, such B cells can be obtained from a human immunizedwith Aβ, fragments, longer polypeptides containing Aβ or fragments oranti-idiotypic antibodies. Optionally, such B cells are obtained from apatient who is ultimately to receive antibody treatment. Antibodiesbinding to Aβ or a fragment thereof are selected. Sequences encodingsuch antibodies (or a binding fragments) are then cloned and amplified.The protocol described by Huse is rendered more efficient in combinationwith phage-display technology. See, e.g., Dower et al., WO 91/17271,McCafferty et al., WO 92/01047, Herzig et al., U.S. Pat. No. 5,877,218,Winter et al., U.S. Pat. No. 5,871,907, Winter et al., U.S. Pat. No.5,858,657, Holliger et al., U.S. Pat. No. 5,837,242, Johnson et al.,U.S. Pat. No. 5,733,743 and Hoogenboom et al., U.S. Pat. No. 5,565,332(each of which is incorporated by reference in its entirety for allpurposes). In these methods, libraries of phage are produced in whichmembers display different antibodies on their outer surfaces. Antibodiesare usually displayed as Fv or Fab fragments. Phage displayingantibodies with a desired specificity are selected by affinityenrichment to an Aβ peptide or fragment thereof.

In a variation of the phage-display method, human antibodies having thebinding specificity of a selected murine antibody can be produced. SeeWinter, WO 92/20791. In this method, either the heavy or light chainvariable region of the selected murine antibody is used as a startingmaterial. If, for example, a light chain variable region is selected asthe starting material, a phage library is constructed in which membersdisplay the same light chain variable region (i.e., the murine startingmaterial) and a different heavy chain variable region. The heavy chainvariable regions are obtained from a library of rearranged human heavychain variable regions. A phage showing strong specific binding for Aβ(e.g., at least 10⁸ and preferably at least 10⁹ M⁻¹) is selected. Thehuman heavy chain variable region from this phage then serves as astarting material for constructing a further phage library. In thislibrary, each phage displays the same heavy chain variable region (i.e.,the region identified from the first display library) and a differentlight chain variable region. The light chain variable regions areobtained from a library of rearranged human variable light chainregions. Again, phage showing strong specific binding for Aβ areselected. These phage display the variable regions of completely humananti-Aβ antibodies. These antibodies usually have the same or similarepitope specificity as the murine starting material.

4. Production of Variable Regions

Having conceptually selected the CDR and framework components ofhumanized immunoglobulins, a variety of methods are available forproducing such immunoglobulins. Because of the degeneracy of the code, avariety of nucleic acid sequences will encode each immunoglobulin aminoacid sequence. The desired nucleic acid sequences can be produced by denovo solid-phase DNA synthesis or by PCR mutagenesis of an earlierprepared variant of the desired polynucleotide. Oligonucleotide-mediatedmutagenesis is a preferred method for preparing substitution, deletionand insertion variants of target polypeptide DNA. See Adelman et al.,DNA 2:183 (1983). Briefly, the target polypeptide DNA is altered byhybridizing an oligonucleotide encoding the desired mutation to asingle-stranded DNA template. After hybridization, a DNA polymerase isused to synthesize an entire second complementary strand of the templatethat incorporates the oligonucleotide primer, and encodes the selectedalteration in the target polypeptide DNA.

5. Selection of Constant Regions

The variable segments of antibodies produced as described supra (e.g.,the heavy and light chain variable regions of chimeric, humanized, orhuman antibodies) are typically linked to at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Human constant region DNA sequences can be isolated inaccordance with well known procedures from a variety of human cells, butpreferably immortalized B cells (see Kabat et al., supra, and Liu etal., WO 87/02671) (each of which is incorporated by reference in itsentirety for all purposes). Ordinarily, the antibody will contain bothlight chain and heavy chain constant regions. The heavy chain constantregion usually includes CH1, hinge, CH2, CH3, and CH4 regions. Theantibodies described herein include antibodies having all types ofconstant regions, including IgM, IgG, IgD, IgA and IgE, and any isotype,including IgG1, IgG2, IgG3 and IgG4. The choice of constant regiondepends, in part, whether antibody-dependent complement and/or cellularmediated toxicity is desired. For example, isotopes IgG1 and IgG3 havecomplement activity and isotypes IgG2 and IgG4 do not. When it isdesired that the antibody (e.g., humanized antibody) exhibit cytotoxicactivity, the constant domain is usually a complement fixing constantdomain and the class is typically IgG1. When such cytotoxic activity isnot desirable, the constant domain may be of the IgG2 class. Choice ofisotype can also affect passage of antibody into the brain. Humanisotype IgG1 is preferred. Light chain constant regions can be lambda orkappa. The humanized antibody may comprise sequences from more than oneclass or isotype. Antibodies can be expressed as tetramers containingtwo light and two heavy chains, as separate heavy chains, light chains,as Fab, Fab′ F(ab′)2, and Fv, or as single chain antibodies in whichheavy and light chain variable domains are linked through a spacer.

6. Expression of Recombinant Antibodies

Chimeric, humanized and human antibodies are typically produced byrecombinant expression. Nucleic acids encoding light and heavy chainvariable regions, optionally linked to constant regions, are insertedinto expression vectors. The light and heavy chains can be cloned in thesame or different expression vectors. The DNA segments encodingimmunoglobulin chains are operably linked to control sequences in theexpression vector(s) that ensure the expression of immunoglobulinpolypeptides. Expression control sequences include, but are not limitedto, promoters (e.g., naturally-associated or heterologous promoters),signal sequences, enhancer elements, and transcription terminationsequences. Preferably, the expression control sequences are eukaryoticpromoter systems in vectors capable of transforming or transfectingeukaryotic host cells. Once the vector has been incorporated into theappropriate host, the host is maintained under conditions suitable forhigh level expression of the nucleotide sequences, and the collectionand purification of the crossreacting antibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers (e.g.,ampicillin-resistance, hygromycin-resistance, tetracycline resistance orneomycin resistance) to permit detection of those cells transformed withthe desired DNA sequences (see, e.g., Itakura et al., U.S. Pat. No.4,704,362).

E. coli is one prokaryotic host particularly useful for cloning thepolynucleotides (e.g., DNA sequences) of the present invention. Othermicrobial hosts suitable for use include bacilli, such as Bacillussubtilus, and other enterobacteriaceae, such as Salmonella, Serratia,and various Pseudomonas species. In these prokaryotic hosts, one canalso make expression vectors, which will typically contain expressioncontrol sequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

Other microbes, such as yeast, are also useful for expression.Saccharomyces is a preferred yeast host, with suitable vectors havingexpression control sequences (e.g., promoters), an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

In addition to microorganisms, mammalian tissue cell culture may also beused to express and produce the polypeptides of the present invention(e.g., polynucleotides encoding immunoglobulins or fragments thereof).See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987).Eukaryotic cells are actually preferred, because a number of suitablehost cell lines capable of secreting heterologous proteins (e.g., intactimmunoglobulins) have been developed in the art, and include CHO celllines, various Cos cell lines, HeLa cells, preferably, myeloma celllines, or transformed B-cells or hybridomas. Preferably, the cells arenonhuman. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, and anenhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, SV40, adenovirus, bovine papilloma virus,cytomegalovirus and the like. See Co et al., J. Immunol. 148:1149(1992).

Alternatively, antibody-coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,Deboer et al., U.S. Pat. No. 5,741,957, Rosen, U.S. Pat. No. 5,304,489,and Meade et al., U.S. Pat. No. 5,849,992). Suitable transgenes includecoding sequences for light and/or heavy chains in operable linkage witha promoter and enhancer from a mammary gland specific gene, such ascasein or beta lactoglobulin.

The vectors containing the polynucleotide sequences of interest (e.g.,the heavy and light chain encoding sequences and expression controlsequences) can be transferred into the host cell by well-known methods,which vary depending on the type of cellular host. For example, calciumchloride transfection is commonly utilized for prokaryotic cells,whereas calcium phosphate treatment, electroporation, lipofection,biolistics or viral-based transfection may be used for other cellularhosts. (See generally Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Press, 2nd ed., 1989) (incorporated byreference in its entirety for all purposes). Other methods used totransform mammalian cells include the use of polybrene, protoplastfusion, liposomes, electroporation, and microinjection (see generally,Sambrook et al., supra). For production of transgenic animals,transgenes can be microinjected into fertilized oocytes, or can beincorporated into the genome of embryonic stem cells, and the nuclei ofsuch cells transferred into enucleated oocytes.

When heavy and light chains are cloned on separate expression vectors,the vectors are co-transfected to obtain expression and assembly ofintact immunoglobulins. Once expressed, the whole antibodies, theirdimers, individual light and heavy chains, or other immunoglobulin formsof the present invention can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns, column chromatography, HPLC purification, gelelectrophoresis and the like (see generally Scopes, Protein Purification(Springer-Verlag, N.Y., (1982)). Substantially pure immunoglobulins ofat least about 90 to 95% homogeneity are preferred, and 98 to 99% ormore homogeneity most preferred, for pharmaceutical uses.

7. Antibody Fragments

Also contemplated within the scope of the instant invention are antibodyfragments. In one embodiment, fragments of non-human, chimeric and/orhuman antibodies are provided. In another embodiment, fragments ofhumanized antibodies are provided. Typically, these fragments exhibitspecific binding to antigen with an affinity of at least 10⁷, and moretypically 10⁸ or 10⁹ M⁻¹. Humanized antibody fragments include separateheavy chains, light chains Fab, Fab′ F(ab′)2, Fabc, and Fv. Fragmentsare produced by recombinant DNA techniques, or by enzymatic or chemicalseparation of intact immunoglobulins.

8. Testing Antibodies for Therapeutic Efficacy in Animal Models

Groups of 7-9 month old PDAPP mice each are injected with 0.5 mg in PBSof polyclonal anti-Aβ or specific anti-Aβ monoclonal antibodies. Allantibody preparations are purified to have low endotoxin levels.Monoclonals can be prepared against a fragment by injecting the fragmentor longer form of Aβ into a mouse, preparing hybridomas and screeningthe hybridomas for an antibody that specifically binds to a desiredfragment of Aβ without binding to other nonoverlapping fragments of Aβ.

Mice are injected intraperitoneally as needed over a 4 month period tomaintain a circulating antibody concentration measured by ELISA titer ofgreater than 1/1000 defined by ELISA to Aβ42 or other immunogen. Titersare monitored and mice are euthanized at the end of 6 months ofinjections. Histochemistry, Aβ levels and toxicology are performed postmortem. Ten mice are used per group.

9. Screening Antibodies for Clearing Activity

The invention also provides methods of screening an antibody foractivity in clearing an amyloid deposit or any other antigen, orassociated biological entity, for which clearing activity is desired. Toscreen for activity against an amyloid deposit, a tissue sample from abrain of a patient with Alzheimer's disease or an animal model havingcharacteristic Alzheimer's pathology is contacted with phagocytic cellsbearing an Fc receptor, such as microglial cells, and the antibody undertest in a medium in vitro. The phagocytic cells can be a primary cultureor a cell line, such as BV-2, C8-B4, or THP-1. In some methods, thecomponents are combined on a microscope slide to facilitate microscopicmonitoring. In some methods, multiple reactions are performed inparallel in the wells of a microtiter dish. In such a format, a separateminiature microscope slide can be mounted in the separate wells, or anonmicroscopic detection format, such as ELISA detection of Aβ can beused. Preferably, a series of measurements is made of the amount ofamyloid deposit in the in vitro reaction mixture, starting from abaseline value before the reaction has proceeded, and one or more testvalues during the reaction. The antigen can be detected by staining, forexample, with a fluorescently labeled antibody to Aβ or other componentof amyloid plaques. The antibody used for staining may or may not be thesame as the antibody being tested for clearing activity. A reductionrelative to baseline during the reaction of the amyloid depositsindicates that the antibody under test has clearing activity. Suchantibodies are likely to be useful in preventing or treating Alzheimer'sand other amyloidogenic diseases.

Analogous methods can be used to screen antibodies for activity inclearing other types of biological entities. The assay can be used todetect clearing activity against virtually any kind of biologicalentity. Typically, the biological entity has some role in human oranimal disease. The biological entity can be provided as a tissue sampleor in isolated form. If provided as a tissue sample, the tissue sampleis preferably unfixed to allow ready access to components of the tissuesample and to avoid perturbing the conformation of the componentsincidental to fixing. Examples of tissue samples that can be tested inthis assay include cancerous tissue, precancerous tissue, tissuecontaining benign growths such as warts or moles, tissue infected withpathogenic microorganisms, tissue infiltrated with inflammatory cells,tissue bearing pathological matrices between cells (e.g., fibrinouspericarditis), tissue bearing aberrant antigens, and scar tissue.Examples of isolated biological entities that can be used include Aβ,viral antigens or viruses, proteoglycans, antigens of other pathogenicmicroorganisms, tumor antigens, and adhesion molecules. Such antigenscan be obtained from natural sources, recombinant expression or chemicalsynthesis, among other means. The tissue sample or isolated biologicalentity is contacted with phagocytic cells bearing Fc receptors, such asmonocytes or microglial cells, and an antibody to be tested in a medium.The antibody can be directed to the biological entity under test or toan antigen associated with the entity. In the latter situation, theobject is to test whether the biological entity is vicariouslyphagocytosed with the antigen. Usually, although not necessarily, theantibody and biological entity (sometimes with an associated antigen),are contacted with each other before adding the phagocytic cells. Theconcentration of the biological entity and/or the associated antigenremaining in the medium, if present, is then monitored. A reduction inthe amount or concentration of antigen or the associated biologicalentity in the medium indicates the antibody has a clearing responseagainst the antigen and/or associated biological entity in conjunctionwith the phagocytic cells (see, e.g., Example IV).

10. Chimeric/Humanized Antibodies Having Altered Effector Function

For the above-described antibodies of the invention comprising aconstant region (Fc region), it may also be desirable to alter theeffector function of the molecule. Generally, the effector function ofan antibody resides in the constant or Fc region of the molecule whichcan mediate binding to various effector molecules, e.g., complementproteins or Fc receptors. The binding of complement to the Fc region isimportant, for example, in the opsonization and lysis of cell pathogensand the activation of inflammatory responses. The binding of antibody toFc receptors, for example, on the surface of effector cells can triggera number of important and diverse biological responses including, forexample, engulfment and destruction of antibody-coated pathogens orparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (i.e., antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer of antibodies, and control of immunoglobulin production.

Accordingly, depending on a particular therapeutic or diagnosticapplication, the above-mentioned immune functions, or only selectedimmune functions, may be desirable. By altering the Fc region of theantibody, various aspects of the effector function of the molecule,including enhancing or suppressing various reactions of the immunesystem, with beneficial effects in diagnosis and therapy, are achieved.

The antibodies of the invention can be produced which react only withcertain types of Fc receptors, for example, the antibodies of theinvention can be modified to bind to only certain Fc receptors, or ifdesired, lack Fc receptor binding entirely, by deletion or alteration ofthe Fc receptor binding site located in the Fc region of the antibody.Other desirable alterations of the Fc region of an antibody of theinvention are cataloged below. Typically the Kabat numbering system isused to indicate which amino acid residue(s) of the Fc region (e.g., ofan IgG antibody) are altered (e.g., by amino acid substitution) in orderto achieve a desired change in effector function. The numbering systemis also employed to compare antibodies across species such that adesired effector function observed in, for example, a mouse antibody,can then be systematically engineered into a human, humanized, orchimeric antibody of the invention.

For example, it has been observed that antibodies (e.g., IgG antibodies)can be grouped into those found to exhibit tight, intermediate, or weakbinding to an Fc receptor (e.g., an Fc receptor on human monocytes(FcγRI)). By comparison of the amino-acid sequences in these differentaffinity groups, a monocyte-binding site in the hinge-link region(Leu234-Ser239) has been identified. Moreover, the human FcγRI receptorbinds human IgG1 and mouse IgG2a as a monomer, but the binding of mouseIgG2b is 100-fold weaker. A comparison of the sequence of these proteinsin the hinge-link region shows that the sequence 234 to 238, i.e.,Leu-Leu-Gly-Gly-Pro in the strong binders becomes Leu-Glu-Gly-Gly-Pro inmouse gamma 2b, i.e., weak binders. Accordingly, a corresponding changein a human antibody hinge sequence can be made if reduced FcγI receptorbinding is desired. It is understood that other alterations can be madeto achieve the same or similar results. For example, the affinity ofFcγRI binding can be altered by replacing the specified residue with aresidue having an inappropriate functional group on its sidechain, or byintroducing a charged functional group (e.g., Glu or Asp) or for examplean aromatic non-polar residue (e.g., Phe, Tyr, or Trp).

These changes can be equally applied to the murine, human, and ratsystems given the sequence homology between the differentimmunoglobulins. It has been shown that for human IgG3, which binds tothe human FcγRI receptor, changing Leu 235 to Glu destroys theinteraction of the mutant for the receptor. The binding site for thisreceptor can thus be switched on or switched off by making theappropriate mutation.

Mutations on adjacent or close sites in the hinge link region (e.g.,replacing residues 234, 236 or 237 by Ala) indicate that alterations inresidues 234, 235, 236, and 237 at least affect affinity for the FcγRIreceptor. Accordingly, the antibodies of the invention can also have analtered Fc region with altered binding affinity for FcγRI as comparedwith the unmodified antibody. Such an antibody conveniently has amodification at amino acid residue 234, 235, 236, or 237.

Affinity for other Fc receptors can be altered by a similar approach,for controlling the immune response in different ways.

As a further example, the lytic properties of IgG antibodies followingbinding of the Cl component of complement can be altered.

The first component of the complement system, Cl, comprises threeproteins known as Clq, Clr and Cls which bind tightly together. It hasbeen shown that Clq is responsible for binding of the three proteincomplex to an antibody.

Accordingly, the Clq binding activity of an antibody can be altered byproviding an antibody with an altered CH 2 domain in which at least oneof the amino acid residues 318, 320, and 322 of the heavy chain has beenchanged to a residue having a different side chain. The numbering of theresidues in the heavy chain is that of the EU index (see Kabat et al.,supra). Other suitable alterations for altering, e.g., reducing orabolishing specific Clq-binding to an antibody include changing any oneof residues 318 (Glu), 320 (Lys) and 322 (Lys), to Ala.

Moreover, by making mutations at these residues, it has been shown thatClq binding is retained as long as residue 318 has a hydrogen-bondingside chain and residues 320 and 322 both have a positively charged sidechain.

Clq binding activity can be abolished by replacing any one of the threespecified residues with a residue having an inappropriate functionalityon its side chain. It is not necessary to replace the ionic residuesonly with Ala to abolish Clq binding. It is also possible to use otheralkyl-substituted non-ionic residues, such as Gly, Ile, Leu, or Val, orsuch aromatic non-polar residues as Phe, Tyr, Trp and Pro in place ofany one of the three residues in order to abolish Clq binding. Inaddition, it is also be possible to use such polar non-ionic residues asSer, Thr, Cys, and Met in place of residues 320 and 322, but not 318, inorder to abolish Clq binding activity.

It is also noted that the side chains on ionic or non-ionic polarresidues will be able to form hydrogen bonds in a similar manner to thebonds formed by the Glu residue. Therefore, replacement of the 318 (Glu)residue by a polar residue may modify but not abolish Clq bindingactivity.

It is also known that replacing residue 297 (Asn) with Ala results inremoval of lytic activity while only slightly reducing (about three foldweaker) affinity for Clq. This alteration destroys the glycosylationsite and the presence of carbohydrate that is required for complementactivation. Any other substitution at this site will also destroy theglycosylation site.

The invention also provides an antibody having an altered effectorfunction wherein the antibody has a modified hinge region. The modifiedhinge region may comprise a complete hinge region derived from anantibody of different antibody class or subclass from that of the CH1domain. For example, the constant domain (CH1) of a class IgG antibodycan be attached to a hinge region of a class IgG4 antibody.Alternatively, the new hinge region may comprise part of a natural hingeor a repeating unit in which each unit in the repeat is derived from anatural hinge region. In one example, the natural hinge region isaltered by converting one or more cysteine residues into a neutralresidue, such as alanine, or by converting suitably placed residues intocysteine residues. Such alterations are carried out using art recognizedprotein chemistry and, preferably, genetic engineering techniques, asdescribed herein.

In one embodiment of the invention, the number of cysteine residues inthe hinge region of the antibody is reduced, for example, to onecysteine residue. This modification has the advantage of facilitatingthe assembly of the antibody, for example, bispecific antibody moleculesand antibody molecules wherein the Fc portion has been replaced by aneffector or reporter molecule, since it is only necessary to form asingle disulfide bond. This modification also provides a specific targetfor attaching the hinge region either to another hinge region or to aneffector or reporter molecule, either directly or indirectly, forexample, by chemical means.

Conversely, the number of cysteine residues in the hinge region of theantibody is increased, for example, at least one more than the number ofnormally occurring cysteine residues. Increasing the number of cysteineresidues can be used to stabilize the interactions between adjacenthinges. Another advantage of this modification is that it facilitatesthe use of cysteine thiol groups for attaching effector or reportermolecules to the altered antibody, for example, a radiolabel.

Accordingly, the invention provides for an exchange of hinge regionsbetween antibody classes, in particular, IgG classes, and/or an increaseor decrease in the number of cysteine residues in the hinge region inorder to achieve an altered effector function (see for example U.S. Pat.No. 5,677,425 which is expressly incorporated herein). A determinationof altered antibody effector function is made using the assays describedherein or other art recognized techniques.

Importantly, the resultant antibody can be subjected to one or moreassays to evaluate any change in biological activity compared to thestarting antibody. For example, the ability of the antibody with analtered Fc region to bind complement or Fc receptors can be assessedusing the assays disclosed herein as well as any art recognized assay.

Production of the antibodies of the invention is carried out by anysuitable technique including techniques described herein as well astechniques known to those skilled in the art. For example an appropriateprotein sequence, e.g. forming part of or all of a relevant constantdomain, e.g., Fc region, i.e., CH2, and/or CH3 domain(s), of anantibody, and include appropriately altered residue(s) can besynthesized and then chemically joined into the appropriate place in anantibody molecule.

Preferably, genetic engineering techniques are used for producing analtered antibody. Preferred techniques include, for example, preparingsuitable primers for use in polymerase chain reaction (PCR) such that aDNA sequence which encodes at least part of an IgG heavy chain, e.g., anFc or constant region (e.g., CH2, and/or CH3) is altered, at one or moreresidues. The segment can then be operably linked to the remainingportion of the antibody, e.g., the variable region of the antibody andrequired regulatory elements for expression in a cell.

The present invention also includes vectors used to transform the cellline, vectors used in producing the transforming vectors, cell linestransformed with the transforming vectors, cell lines transformed withpreparative vectors, and methods for their production.

Preferably, the cell line which is transformed to produce the antibodywith an altered Fc region (i.e., of altered effector function) is animmortalized mammalian cell line (e.g., CHO cell).

Although the cell line used to produce the antibody with an altered Fcregion is preferably a mammalian cell line, any other suitable cellline, such as a bacterial cell line or a yeast cell line, mayalternatively be used.

B. Nucleic Acid Encoding Immunologic and Therapeutic Agents

Immune responses against amyloid deposits can also be induced byadministration of nucleic acids encoding antibodies and their componentchains used for passive immunization. Such nucleic acids can be DNA orRNA. A nucleic acid segment encoding an immunogen is typically linked toregulatory elements, such as a promoter and enhancer, that allowexpression of the DNA segment in the intended target cells of a patient.For expression in blood cells, as is desirable for induction of animmune response, promoter and enhancer elements from light or heavychain immunoglobulin genes or the CMV major intermediate early promoterand enhancer are suitable to direct expression. The linked regulatoryelements and coding sequences are often cloned into a vector. Foradministration of double-chain antibodies, the two chains can be clonedin the same or separate vectors.

A number of viral vector systems are available including retroviralsystems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop.3:102-109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol.67:5911 (1993)); adeno-associated virus vectors (see, e.g., Zhou et al.,J. Exp. Med. 179:1867 (1994)), viral vectors from the pox familyincluding vaccinia virus and the avian pox viruses, viral vectors fromthe alpha virus genus such as those derived from Sindbis and SemlikiForest Viruses (see, e.g., Dubensky et al., J. Virol. 70:508 (1996)),Venezuelan equine encephalitis virus (see Johnston et al., U.S. Pat. No.5,643,576) and rhabdoviruses, such as vesicular stomatitis virus (seeRose, WO 96/34625) and papillomaviruses (Ohe et al., Human Gene Therapy6:325 (1995); Woo et al., WO 94/12629 and Xiao & Brandsma, NucleicAcids. Res. 24, 2630-2622 (1996)).

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by Eppstein et al., U.S. Pat. No. 5,208,036, Felgner et al.,U.S. Pat. No. 5,264,618, Rose, U.S. Pat. No. 5,279,833, and Epand etal., U.S. Pat. No. 5,283,185. Vectors and DNA encoding an immunogen canalso be adsorbed to or associated with particulate carriers, examples ofwhich include polymethyl methacrylate polymers and polylactides and poly(lactide-co-glycolides), see, e.g., McGee et al., J. Micro Encap.(1996).

Gene therapy vectors or naked polypeptides (e.g., DNA) can be deliveredin vivo by administration to an individual patient, typically bysystemic administration (e.g., intravenous, intraperitoneal, nasal,gastric, intradermal, intramuscular, subdermal, or intracranialinfusion) or topical application (see e.g., Anderson et al., U.S. Pat.No. 5,399,346). The term “naked polynucleotide” refers to apolynucleotide not complexed with colloidal materials. Nakedpolynucleotides are sometimes cloned in a plasmid vector. Such vectorscan further include facilitating agents such as bupivacine (Attardo etal., U.S. Pat. No. 5,593,970). DNA can also be administered using a genegun. See Xiao & Brandsma, supra. The DNA encoding an immunogen isprecipitated onto the surface of microscopic metal beads. Themicroprojectiles are accelerated with a shock wave or expanding heliumgas, and penetrate tissues to a depth of several cell layers. Forexample, The Accel™ Gene Delivery Device manufactured by Agacetus, Inc.Middleton Wis. is suitable. Alternatively, naked DNA can pass throughskin into the blood stream simply by spotting the DNA onto skin withchemical or mechanical irritation (see Howell et al., WO 95/05853).

In a further variation, vectors encoding immunogens can be delivered tocells ex vivo, such as cells explanted from an individual patient (e.g.,lymphocytes, bone marrow aspirates, tissue biopsy) or universal donorhematopoietic stem cells, followed by reimplantation of the cells into apatient, usually after selection for cells which have incorporated thevector.

II. Prophylactic and Therapeutic Methods

The present invention is directed inter alia to treatment of Alzheimer'sand other amyloidogenic diseases by administration of therapeuticimmunological reagents (e.g., humanized immunoglobulins) to specificepitopes within Aβ to a patient under conditions that generate abeneficial therapeutic response in a patient (e.g., induction ofphagocytosis of Aβ, reduction of plaque burden, inhibition of plaqueformation, reduction of neuritic dystrophy, improving cognitivefunction, and/or reversing, treating or preventing cognitive decline) inthe patient, for example, for the prevention or treatment of anamyloidogenic disease. The invention is also directed to use of thedisclosed immunological reagents (e.g., humanized immunoglobulins) inthe manufacture of a medicament for the treatment or prevention of anamyloidogenic disease.

The term “treatment” as used herein, is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease.

In one aspect, the invention provides methods of preventing or treatinga disease associated with amyloid deposits of Aβ in the brain of apatient. Such diseases include Alzheimer's disease, Down's syndrome andcognitive impairment. The latter can occur with or without othercharacteristics of an amyloidogenic disease. Some methods of theinvention entail administering an effective dosage of an antibody thatspecifically binds to a component of an amyloid deposit to the patient.Such methods are particularly useful for preventing or treatingAlzheimer's disease in human patients. Exemplary methods entailadministering an effective dosage of an antibody that binds to Aβ.Preferred methods entail administering an effective dosage of anantibody that specifically binds to an epitope within residues 1-10 ofAβ, for example, antibodies that specifically bind to an epitope withinresidues 1-3 of Aβ, antibodies that specifically bind to an epitopewithin residues 1-4 of Aβ, antibodies that specifically bind to anepitope within residues 1-5 of Aβ, antibodies that specifically bind toan epitope within residues 1-6 of Aβ, antibodies that specifically bindto an epitope within residues 1-7 of Aβ, or antibodies that specificallybind to an epitope within residues 3-7 of Aβ. In yet another aspect, theinvention features administering antibodies that bind to an epitopecomprising a free N-terminal residue of Aβ. In yet another aspect, theinvention features administering antibodies that bind to an epitopewithin residues of 1-10 of Aβ wherein residue 1 and/or residue 7 of Aβis aspartic acid. In yet another aspect, the invention featuresadministering antibodies that specifically bind to Aβ peptide withoutbinding to full-length amyloid precursor protein (APP). In yet anotheraspect, the isotype of the antibody is human IgG1.

In yet another aspect, the invention features administering antibodiesthat bind to an amyloid deposit in the patient and induce a clearingresponse against the amyloid deposit. For example, such a clearingresponse can be effected by Fc receptor mediated phagocytosis.

Therapeutic agents of the invention are typically substantially purefrom undesired contaminant. This means that an agent is typically atleast about 50% w/w (weight/weight) purity, as well as beingsubstantially free from interfering proteins and contaminants. Sometimesthe agents are at least about 80% w/w and, more preferably at least 90or about 95% w/w purity. However, using conventional proteinpurification techniques, homogeneous peptides of at least 99% w/w can beobtained.

The methods can be used on both asymptomatic patients and thosecurrently showing symptoms of disease. The antibodies used in suchmethods can be human, humanized, chimeric or nonhuman antibodies, orfragments thereof (e.g., antigen binding fragments) and can bemonoclonal or polyclonal, as described herein. In yet another aspect,the invention features administering antibodies prepared from a humanimmunized with Aβ peptide, which human can be the patient to be treatedwith antibody.

In another aspect, the invention features administering an antibody witha pharmaceutical carrier as a pharmaceutical composition. Alternatively,the antibody can be administered to a patient by administering apolynucleotide encoding at least one antibody chain. The polynucleotideis expressed to produce the antibody chain in the patient. Optionally,the polynucleotide encodes heavy and light chains of the antibody. Thepolynucleotide is expressed to produce the heavy and light chains in thepatient. In exemplary embodiments, the patient is monitored for level ofadministered antibody in the blood of the patient.

The invention thus fulfills a longstanding need for therapeutic regimesfor preventing or ameliorating the neuropathology and, in some patients,the cognitive impairment associated with Alzheimer's disease.

A. Patients Amenable to Treatment

Patients amenable to treatment include individuals at risk of diseasebut not showing symptoms, as well as patients presently showingsymptoms. In the case of Alzheimer's disease, virtually anyone is atrisk of suffering from Alzheimer's disease if he or she lives longenough. Therefore, the present methods can be administeredprophylactically to the general population without the need for anyassessment of the risk of the subject patient. The present methods areespecially useful for individuals who have a known genetic risk ofAlzheimer's disease. Such individuals include those having relatives whohave experienced this disease, and those whose risk is determined byanalysis of genetic or biochemical markers. Genetic markers of risktoward Alzheimer's disease include mutations in the APP gene,particularly mutations at position 717 and positions 670 and 671referred to as the Hardy and Swedish mutations respectively (see Hardy,supra). Other markers of risk are mutations in the presenilin genes, PS1and PS2, and ApoE4, family history of AD, hypercholesterolemia oratherosclerosis. Individuals presently suffering from Alzheimer'sdisease can be recognized from characteristic dementia, as well as thepresence of risk factors described above. In addition, a number ofdiagnostic tests are available for identifying individuals who have AD.These include measurement of CSF tau and Aβ42 levels. Elevated tau anddecreased Aβ42 levels signify the presence of AD. Individuals sufferingfrom Alzheimer's disease can also be diagnosed by ADRDA criteria asdiscussed in the Examples section.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70. Treatment typically entails multipledosages over a period of time. Treatment can be monitored by assayingantibody levels over time. If the response falls, a booster dosage isindicated. In the case of potential Down's syndrome patients, treatmentcan begin antenatally by administering therapeutic agent to the motheror shortly after birth.

B. Treatment Regimes and Dosages

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a patient susceptible to, or otherwise at risk of,Alzheimer's disease in an amount sufficient to eliminate or reduce therisk, lessen the severity, or delay the outset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. In therapeutic applications, compositions ormedicants are administered to a patient suspected of, or alreadysuffering from such a disease in an amount sufficient to cure, or atleast partially arrest, the symptoms of the disease (biochemical,histologic and/or behavioral), including its complications andintermediate pathological phenotypes in development of the disease.

In some methods, administration of agent reduces or eliminatesmyocognitive impairment in patients that have not yet developedcharacteristic Alzheimer's pathology. An amount adequate to accomplishtherapeutic or prophylactic treatment is defined as a therapeutically-or prophylactically-effective dose. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until asufficient immune response has been achieved. The term “immune response”or “immunological response” includes the development of a humoral(antibody mediated) and/or a cellular (mediated by antigen-specific Tcells or their secretion products) response directed against an antigenin a recipient subject. Such a response can be an active response, i.e.,induced by administration of immunogen, or a passive response, i.e.,induced by administration of immunoglobulin or antibody or primedT-cells.

An “immunogenic agent” or “immunogen” is capable of inducing animmunological response against itself on administration to a mammal,optionally in conjunction with an adjuvant. Typically, the immuneresponse is monitored and repeated dosages are given if the immuneresponse starts to wane.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnon-human mammals including transgenic mammals can also be treated.Treatment dosages need to be titrated to optimize safety and efficacy.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host bodyweight. For example dosages can be 1 mg/kg body weight or 10 mg/kg bodyweight or within the range of 1-10 mg/kg, preferably at least 1 mg/kg.Subjects can be administered such doses daily, on alternative days,weekly or according to any other schedule determined by empiricalanalysis. An exemplary treatment entails administration in multipledosages over a prolonged period, for example, of at least six months.Additional exemplary treatment regimes entail administration once perevery two weeks or once a month or once every 3 to 6 months. Exemplarydosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or moremonoclonal antibodies with different binding specificities areadministered simultaneously, in which case the dosage of each antibodyadministered falls within the ranges indicated.

Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. Intervals canalso be irregular as indicated by measuring blood levels of antibody toAβ in the patient. In some methods, dosage is adjusted to achieve aplasma antibody concentration of 1-1000 μg/ml and in some methods 25-300μg/ml. Alternatively, antibody can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of theantibody in the patient. In general, human antibodies show the longesthalf-life, followed by humanized antibodies, chimeric antibodies, andnonhuman antibodies.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, compositions containing the present antibodies or acocktail thereof are administered to a patient not already in thedisease state to enhance the patient's resistance. Such an amount isdefined to be a “prophylactic effective dose.” In this use, the preciseamounts again depend upon the patient's state of health and generalimmunity, but generally range from 0.1 to 25 mg per dose, especially 0.5to 2.5 mg per dose. A relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives.

In therapeutic applications, a relatively high dosage (e.g., from about1 to 200 mg of antibody per dose, with dosages of from 5 to 25 mg beingmore commonly used) at relatively short intervals is sometimes requireduntil progression of the disease is reduced or terminated, andpreferably until the patient shows partial or complete amelioration ofsymptoms of disease. Thereafter, the patent can be administered aprophylactic regime.

Doses for nucleic acids encoding antibodies range from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Dosesfor infectious viral vectors vary from 10-100, or more, virions perdose.

Therapeutic agents can be administered by parenteral, topical,intravenous, oral, subcutaneous, intraarterial, intracranial,intraperitoneal, intranasal or intramuscular means for prophylacticand/or therapeutic treatment. The most typical route of administrationof an immunogenic agent is subcutaneous although other routes can beequally effective. The next most common route is intramuscularinjection. This type of injection is most typically performed in the armor leg muscles. In some methods, agents are injected directly into aparticular tissue where deposits have accumulated, for exampleintracranial injection. Intramuscular injection or intravenous infusionare preferred for administration of antibody. In some methods,particular therapeutic antibodies are injected directly into thecranium. In some methods, antibodies are administered as a sustainedrelease composition or device, such as a Medipad™ device.

Agents of the invention can optionally be administered in combinationwith other agents that are at least partly effective in treatment ofamyloidogenic disease. In the case of Alzheimer's and Down's syndrome,in which amyloid deposits occur in the brain, agents of the inventioncan also be administered in conjunction with other agents that increasepassage of the agents of the invention across the blood-brain barrier.

C. Pharmaceutical Compositions

Agents of the invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent, i.e., and a varietyof other pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.(1980)). The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions can alsoinclude, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

For parenteral administration, agents of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.Antibodies can be administered in the form of a depot injection orimplant preparation, which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises monoclonal antibody at 5 mg/mL, formulated inaqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (see Langer, Science 249:1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28:97 (1997)). Theagents of this invention can be administered in the form of a depotinjection or implant preparation, which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications. For suppositories, binders and carriersinclude, for example, polyalkylene glycols or triglycerides; suchsuppositories can be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1%-2%. Oralformulations include excipients, such as pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, and magnesium carbonate. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10%-95% of active ingredient,preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391, 851(1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin path orusing transferosomes (Paul et al., Eur. J. Immunol. 25:3521 (1995); Cevcet al., Biochem. Biophys. Acta 1368:201-15 (1998)).

III. Monitoring the Course of Treatment

The invention provides methods of monitoring treatment in a patientsuffering from or susceptible to Alzheimer's, i.e., for monitoring acourse of treatment being administered to a patient. The methods can beused to monitor both therapeutic treatment on symptomatic patients andprophylactic treatment on asymptomatic patients. In particular, themethods are useful for monitoring passive immunization (e.g., measuringlevel of administered antibody).

Some methods entail determining a baseline value, for example, of anantibody level or profile in a patient, before administering a dosage ofagent, and comparing this with a value for the profile or level aftertreatment. A significant increase (i.e., greater than the typical marginof experimental error in repeat measurements of the same sample,expressed as one standard deviation from the mean of such measurements)in value of the level or profile signals a positive treatment outcome(i.e., that administration of the agent has achieved a desiredresponse). If the value for immune response does not changesignificantly, or decreases, a negative treatment outcome is indicated.

In other methods, a control value (i.e., a mean and standard deviation)of level or profile is determined for a control population. Typicallythe individuals in the control population have not received priortreatment. Measured values of the level or profile in a patient afteradministering a therapeutic agent are then compared with the controlvalue. A significant increase relative to the control value (e.g.,greater than one standard deviation from the mean) signals a positive orsufficient treatment outcome. A lack of significant increase or adecrease signals a negative or insufficient treatment outcome.Administration of agent is generally continued while the level isincreasing relative to the control value. As before, attainment of aplateau relative to control values is an indicator that theadministration of treatment can be discontinued or reduced in dosageand/or frequency.

In other methods, a control value of the level or profile (e.g., a meanand standard deviation) is determined from a control population ofindividuals who have undergone treatment with a therapeutic agent andwhose levels or profiles have plateaued in response to treatment.Measured values of levels or profiles in a patient are compared with thecontrol value. If the measured level in a patient is not significantlydifferent (e.g., more than one standard deviation) from the controlvalue, treatment can be discontinued. If the level in a patient issignificantly below the control value, continued administration of agentis warranted. If the level in the patient persists below the controlvalue, then a change in treatment may be indicated.

In other methods, a patient who is not presently receiving treatment buthas undergone a previous course of treatment is monitored for antibodylevels or profiles to determine whether a resumption of treatment isrequired. The measured level or profile in the patient can be comparedwith a value previously achieved in the patient after a previous courseof treatment. A significant decrease relative to the previousmeasurement (i.e., greater than a typical margin of error in repeatmeasurements of the same sample) is an indication that treatment can beresumed. Alternatively, the value measured in a patient can be comparedwith a control value (mean plus standard deviation) determined in apopulation of patients after undergoing a course of treatment.Alternatively, the measured value in a patient can be compared with acontrol value in populations of prophylactically treated patients whoremain free of symptoms of disease, or populations of therapeuticallytreated patients who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the control level(i.e., more than a standard deviation) is an indicator that treatmentshould be resumed in a patient.

The tissue sample for analysis is typically blood, plasma, serum, mucousfluid or cerebrospinal fluid from the patient. The sample is analyzed,for example, for levels or profiles of antibodies to Aβ peptide, e.g.,levels or profiles of humanized antibodies. ELISA methods of detectingantibodies specific to Aβ are described in the Examples section. In somemethods, the level or profile of an administered antibody is determinedusing a clearing assay, for example, in an in vitro phagocytosis assay,as described herein. In such methods, a tissue sample from a patientbeing tested is contacted with amyloid deposits (e.g., from a PDAPPmouse) and phagocytic cells bearing Fc receptors. Subsequent clearing ofthe amyloid deposit is then monitored. The existence and extent ofclearing response provides an indication of the existence and level ofantibodies effective to clear Aβ in the tissue sample of the patientunder test.

The antibody profile following passive immunization typically shows animmediate peak in antibody concentration followed by an exponentialdecay. Without a further dosage, the decay approaches pretreatmentlevels within a period of days to months depending on the half-life ofthe antibody administered. For example the half-life of some humanantibodies is of the order of 20 days.

In some methods, a baseline measurement of antibody to Aβ in the patientis made before administration, a second measurement is made soonthereafter to determine the peak antibody level, and one or more furthermeasurements are made at intervals to monitor decay of antibody levels.When the level of antibody has declined to baseline or a predeterminedpercentage of the peak less baseline (e.g., 50%, 25% or 10%),administration of a further dosage of antibody is administered. In somemethods, peak or subsequent measured levels less background are comparedwith reference levels previously determined to constitute a beneficialprophylactic or therapeutic treatment regime in other patients. If themeasured antibody level is significantly less than a reference level(e.g., less than the mean minus one standard deviation of the referencevalue in population of patients benefiting from treatment)administration of an additional dosage of antibody is indicated.

Additional methods include monitoring, over the course of treatment, anyart-recognized physiologic symptom (e.g., physical or mental symptom)routinely relied on by researchers or physicians to diagnose or monitoramyloidogenic diseases (e.g., Alzheimer's disease). For example, one canmonitor cognitive impairment. The latter is a symptom of Alzheimer'sdisease and Down's syndrome but can also occur without othercharacteristics of either of these diseases. For example, cognitiveimpairment can be monitored by determining a patient's score on theMini-Mental State Exam in accordance with convention throughout thecourse of treatment.

C. Kits

The invention further provides kits for performing the monitoringmethods described above. Typically, such kits contain an agent thatspecifically binds to antibodies to Aβ. The kit can also include alabel. For detection of antibodies to Aβ, the label is typically in theform of labeled anti-idiotypic antibodies. For detection of antibodies,the agent can be supplied prebound to a solid phase, such as to thewells of a microtiter dish. Kits also typically contain labelingproviding directions for use of the kit. The labeling may also include achart or other correspondence regime correlating levels of measuredlabel with levels of antibodies to Aβ. The term labeling refers to anywritten or recorded material that is attached to, or otherwiseaccompanies a kit at any time during its manufacture, transport, sale oruse. For example, the term labeling encompasses advertising leaflets andbrochures, packaging materials, instructions, audio or videocassettes,computer discs, as well as writing imprinted directly on kits.

The invention also provides diagnostic kits, for example, research,detection and/or diagnostic kits (e.g., for performing in vivo imaging).Such kits typically contain an antibody for binding to an epitope of Aβ,preferably within residues 1-10. Preferably, the antibody is labeled ora secondary labeling reagent is included in the kit. Preferably, the kitis labeled with instructions for performing the intended application,for example, for performing an in vivo imaging assay. Exemplaryantibodies are those described herein.

D. In Vivo Imaging

The invention provides methods of in vivo imaging amyloid deposits in apatient. Such methods are useful to diagnose or confirm diagnosis ofAlzheimer's disease, or susceptibility thereto. For example, the methodscan be used on a patient presenting with symptoms of dementia. If thepatient has abnormal amyloid deposits, then the patient is likelysuffering from Alzheimer's disease. The methods can also be used onasymptomatic patients. Presence of abnormal deposits of amyloidindicates susceptibility to future symptomatic disease. The methods arealso useful for monitoring disease progression and/or response totreatment in patients who have been previously diagnosed withAlzheimer's disease.

The methods work by administering a reagent, such as antibody that bindsto Aβ, to the patient and then detecting the agent after it has bound.Preferred antibodies bind to Aβ deposits in a patient without binding tofull length APP polypeptide. Antibodies binding to an epitope of Aβwithin amino acids 1-10 are particularly preferred. In some methods, theantibody binds to an epitope within amino acids 7-10 of Aβ. Suchantibodies typically bind without inducing a substantial clearingresponse. In other methods, the antibody binds to an epitope withinamino acids 1-7 of Aβ. Such antibodies typically bind and induce aclearing response to Aβ. However, the clearing response can be avoidedby using antibody fragments lacking a full-length constant region, suchas Fabs. In some methods, the same antibody can serve as both atreatment and diagnostic reagent. In general, antibodies binding toepitopes C-terminal to residue 10 of Aβ do not show as strong a signalas antibodies binding to epitopes within residues 1-10, presumablybecause the C-terminal epitopes are inaccessible in amyloid deposits.Accordingly, such antibodies are less preferred.

Diagnostic reagents can be administered by intravenous injection intothe body of the patient, or directly into the brain by intracranialinjection or by drilling a hole through the skull. The dosage of reagentshould be within the same ranges as for treatment methods. Typically,the reagent is labeled, although in some methods, the primary reagentwith affinity for Aβ is unlabelled and a secondary labeling agent isused to bind to the primary reagent. The choice of label depends on themeans of detection. For example, a fluorescent label is suitable foroptical detection. Use of paramagnetic labels is suitable fortomographic detection without surgical intervention. Radioactive labelscan also be detected using PET or SPECT.

Diagnosis is performed by comparing the number, size, and/or intensityof labeled loci, to corresponding baseline values. The base line valuescan represent the mean levels in a population of undiseased individuals.Baseline values can also represent previous levels determined in thesame patient. For example, baseline values can be determined in apatient before beginning treatment, and measured values thereaftercompared with the baseline values. A decrease in values relative tobaseline signals a positive response to treatment.

The present invention will be more fully described by the followingnon-limiting examples.

EXAMPLES Example I Therapeutic Efficacy of Anti-Aβ Antibodies: mAb 2H3,mAb 10D5, mAb 266, mAb 21F12 and pAb Aβ1-42

This example tests the capacity of various monoclonal and polyclonalantibodies to Aβ to inhibit accumulation of Aβ in the brain ofheterozygotic transgenic mice.

A. Study Design

Sixty male and female, heterozygous PDAPP transgenic mice, 8.5 to 10.5months of age were obtained from Charles River Laboratory. The mice weresorted into six groups to be treated with various antibodies directed toAβ. Animals were distributed to match the gender, age, parentage andsource of the animals within the groups as closely as possible. Table 2depicts the Experimental design. TABLE 2 Experimental Design TreatmentTreatment Antibody Antibody Group N^(a) Antibody Specificity Isotype 1 9none NA^(b) NA (PBS alone) 2 10 Polyclonal Aβ1-42 mixed 3 0 mAb^(d) 2H3Aβ1-12 IgG1 4 8 mAb 10D5 Aβ3-7 IgG1 5 6 mAb 266 Aβ13-28 IgG1 6 8 mAb21F12 Aβ33-42 IgG2a^(a)Number of mice in group at termination of the experiment. All groupsstarted with 10 animals per group.^(b)NA: not applicable^(c)mouse polyclonal: anti-aggregated Aβ42^(d)mAb: monoclonal antibody

As shown in Table 2, the antibodies included four murine Aβ-specificmonoclonal antibodies, 2H3 (directed to Aβ residues 1-12), 10D5(directed to Aβ residues 3-7) (10D5 is produced by the cell linedesignated RB44-10D5.19.21, which was deposited on Apr. 8, 2003 andassigned ATCC accession number PTA-5129), 266 (directed to Aβ residues13-28 and binds to soluble but not to aggregated AN1792), 21F12(directed to Aβ residues 33-42). A fifth group was treated with anAβ-specific polyclonal antibody fraction (raised by immunization withaggregated AN1792). The negative control group received the diluent,PBS, alone without antibody.

B. Monitoring the Course of Treatment

The monoclonal antibodies were injected at a dose of about 10 mg/kg(assuming that the mice weighed 50 g). Antibody titers were monitoredover the 28 weeks of treatment. Injections were administeredintraperitoneally every seven days on average to maintain anti-Aβ titersabove 1000. Although lower titers were measured for mAb 266 since itdoes not bind well to the aggregated AN1792 used as the capture antigenin the assay, the same dosing schedule was maintained for this group.The group receiving monoclonal antibody 2H3 was discontinued within thefirst three weeks since the antibody was cleared too rapidly in vivo.

For determination of antibody titers, a subset of three randomly chosenmice from each group were bled just prior to each intraperitonealinoculation, for a total of 30 bleeds. Antibody titers were measured asAβ1-42-binding antibody using a sandwich ELISA with plastic multi-wellplates coated with Aβ1-42 as described in detail in the GeneralMaterials and Methods. Mean titers for each bleed are set forth in Table3 for the polyclonal antibody and the monoclonals 10D5 and 21F12. TABLE3 weeks weeks weeks 21F12 21F12 10D5 10D5 poly poly 0.15 500 0.15 30000.15 1600 0.5 800 0.5 14000 0.5 4000 1 2500 1 5000 1 4500 1.5 1800 1.15000 1.5 3000 2 1400 1.2 1300 2 1300 3 6000 2 3000 3 1600 3.5 550 3 40003.5 650 4 1600 3.5 500 4 1300 5 925 4 2400 5 450 6 3300 5 925 6 2100 74000 6 1700 7 1300 8 1400 7 1600 8 2300 9 1900 8 4000 9 700 10 1700 91800 10 600 11 1600 10 1800 11 600 12 1000 11 2300 12 1000 13 1500 122100 13 900 14 1300 13 2800 14 1900 15 1000 14 1900 15 1200 16 1700 152700 16 700 17 1700 16 1300 17 2100 18 5000 17 2200 18 1800 19 900 182200 19 1800 20 300 19 2500 20 1200 22 1750 20 980 22 1000 23 1600 222000 23 1200 24 1000 23 1000 24 675 25 1100 24 850 25 850 26 2250 25 60026 1600 27 1400 26 1100 27 1900 28 27 1450 28 28

Titers averaged about 1000 over this time period for the polyclonalantibody preparation and were slightly above this level for the 10D5-and 21F12-treated animals.

Treatment was continued over a six-month period for a total of 196 days.Animals were euthanized one week after the final dose.

C. Aβ and APP Levels in the Brain:

Following about six months of treatment with the various anti-Aβantibody preparations, brains were removed from the animals followingsaline perfusion. One hemisphere was prepared for immunohistochemicalanalysis and the second was used for the quantitation of Aβ and APPlevels. To measure the concentrations of various forms of beta amyloidpeptide and amyloid precursor protein (APP), the hemisphere wasdissected and homogenates of the hippocampal, cortical, and cerebellarregions were prepared in 5M guanidine. These were serially diluted andthe level of amyloid peptide or APP was quantitated by comparison to aseries of dilutions of standards of Aβ peptide or APP of knownconcentrations in an ELISA format.

The levels of total Aβ and of Aβ1-42 measured by ELISA in homogenates ofthe cortex, and the hippocampus and the level of total Aβ in thecerebellum are shown in Tables 4, 5, and 6, respectively. The medianconcentration of total Aβ for the control group, inoculated with PBS,was 3.6-fold higher in the hippocampus than in the cortex (median of63,389 ng/g hippocampal tissue compared to 17,818 ng/g for the cortex).The median level in the cerebellum of the control group (30.6 ng/gtissue) was more than 2,000-fold lower than in the hippocampus. Theselevels are similar to those previously reported for heterozygous PDAPPtransgenic mice of this age (Johnson-Wood et al., supra).

For the cortex, one treatment group had a median Aβ level, measured asAβ1-42, which differed significantly from that of the control group(p<0.05), those animals receiving the polyclonal anti-Aβ antibody asshown in Table 4. The median level of Aβ1-42 was reduced by 65%,compared to the control for this treatment group. The median levels ofAβ1-42 were also significantly reduced by 55% compared to the control inone additional treatment group, those animals dosed with the mAb 10D5(p=0.0433). TABLE 4 CORTEX Medians Means Total Aβ Aβ42 Total Aβ Aβ42Treatment ELISA ELISA ELISA ELISA Group N^(a) value^(b) P value^(c) %Change value P value % Change value value PBS 9 17818 NA^(d) NA 13802 NANA 16150 +/− 7456^(e ) 12621 +/− 5738  Polyclonal anti− 10 6160 0.0055−65 4892 0.0071 −65 5912 +/− 4492 4454 +/− 3347 Aβ42 mAb 10D5 8 79150.1019 −56 6214 0.0433 −55 9695 +/− 6929 6943 +/− 3351 mAb 266 6 91440.1255 −49 8481 0.1255 −39 9204 +/− 9293 7489 +/− 6921 mAb 21F12 8 151580.2898 −15 13578 0.7003  −2 12481 +/− 7082  11005 +/− 6324 Footnotes:^(a)Number of animals per group at the end of the experiment^(b)ng/g tissue^(c)Mann Whitney analysis^(d)NA: not applicable^(e)Standard Deviation

In the hippocampus, the median percent reduction of total Aβ associatedwith treatment with polyclonal anti-Aβ antibody (50%, p=0.0055) was notas great as that observed in the cortex (65%) (Table 5). However, theabsolute magnitude of the reduction was almost 3-fold greater in thehippocampus than in the cortex, a net reduction of 31,683 ng/g tissue inthe hippocampus versus 11,658 ng/g tissue in the cortex. When measuredas the level of the more amyloidogenic form of Aβ, Aβ1-42, rather thanas total Aβ, the reduction achieved with the polyclonal antibody wassignificant (p=0.0025). The median levels in groups treated with themAbs 10D5 and 266 were reduced by 33% and 21%, respectively. TABLE 5HIPPOCAMPUS Medians Means Total Aβ Aβ42 Total Aβ Aβ42 Treatment ELISA P% ELISA P % ELISA ELISA Group N^(a) value^(b) value^(c) Change valuevalue Change value value PBS 9 63389 NA^(d) NA 54429 NA NA  58351 +/−13308^(e) 52801 +/− 14701 Polyclonal 10 31706 0.0055 −50 27127 0.0025−50 30058 +/− 22454 24853 +/− 18262 anti-Aβ42 mAb 10D5 8 46779 0.0675−26 36290 0.0543 −33 44581 +/− 18632 36465 +/− 17146 mAb 266 6 486890.0990 −23 43034 0.0990 −21 36419 +/− 27304 32919 +/− 25372 mAb 21F12 851563 0.7728 −19 47961 0.8099 −12 57327 +/− 28927 50305 +/− 23927^(a)Number of animals per group at the end of the experiment^(b)ng/g tissue^(c)Mann Whitney analysis^(d)NA: not applicable^(e)Standard Deviation

Total Aβ was also measured in the cerebellum (Table 6). Those groupsdosed with the polyclonal anti-Aβ and the 266 antibody showedsignificant reductions of the levels of total Aβ (43% and 46%, p=0.0033and p=0.0184, respectively) and that group treated with 10D5 had a nearsignificant reduction (29%, p=0.0675). TABLE 6 CEREBELLUM Medians MeansTotal Aβ Total Aβ Treatment ELISA ELISA Group N^(a) value^(b) Pvalue^(c) % Change value PBS 9 30.64 NA^(d) NA  40.00 +/− 31.89^(e)Polyclonal 10 17.61 0.0033 −43 18.15 +/− 4.36  anti-Aβ42 mAb 10D5 821.68 0.0675 −29 27.29 +/− 19.43 mAb 266 6 16.59 0.0184 −46 19.59 +/−6.59  mAb 21F12 8 29.80 >0.9999  −3 32.88 +/− 9.90 ^(a)Number of animals per group at the end of the experiment^(b)ng/g tissue^(c)Mann Whitney analysis^(d)NA: not applicable^(e)Standard Deviation

APP concentration was also determined by ELISA in the cortex andcerebellum from antibody-treated and control, PBS-treated mice. Twodifferent APP assays were utilized. The first, designated APP-α/FL,recognizes both APP-alpha (α, the secreted form of APP which has beencleaved within the Aβ sequence), and full-length forms (FL) of APP,while the second recognizes only APP-α. In contrast to thetreatment-associated diminution of Aβ in a subset of treatment groups,the levels of APP were virtually unchanged in all of the treatedcompared to the control animals. These results indicate that theimmunizations with Aβ antibodies deplete Aβ without depleting APP.

In summary, Aβ levels were significantly reduced in the cortex,hippocampus and cerebellum in animals treated with the polyclonalantibody raised against AN1792. To a lesser extent monoclonal antibodiesto the amino terminal region of Aβ1-42, specifically amino acids 1-16and 13-28 also showed significant treatment effects.

D. Histochemical Analyses:

The morphology of Aβ-immunoreactive plaques in subsets of brains frommice in the PBS, polyclonal Aβ42, 21F12, 266 and 10D5 treatment groupswas qualitatively compared to that of previous studies in which standardimmunization procedures with Aβ42 were followed.

The largest alteration in both the extent and appearance of amyloidplaques occurred in the animals immunized with the polyclonal Aβ42antibody. The reduction of amyloid load, eroded plaque morphology andcell-associated Aβ immunoreactivity closely resembled effects producedby the standard immunization procedure. These observations support theELISA results in which significant reductions in both total Aβ and Aβ42were achieved by administration of the polyclonal Aβ42 antibody.

In similar qualitative evaluations, amyloid plaques in the 10D5 groupwere also reduced in number and appearance, with some evidence ofcell-associated Aβ immunoreactivity. Relative to control-treatedanimals, the polyclonal Ig fraction against Aβ and one of the monoclonalantibodies (10D5) reduced plaque burden by 93% and 81%, respectively(p<0.005). 21F12 appeared to have a relatively modest effect on plaqueburden. Micrographs of brain after treatment with pAbAβ₁₋₄₂ show diffusedeposits and absence of many of the larger compacted plaques in thepAbAβ₁₋₄₂ treated group relative to control treated animals.

E. Lymphoproliferative Responses

Aβ-dependent lymphoproliferation was measured using spleen cellsharvested eight days following the final antibody infusion. Freshlyharvested cells, 10⁵ per well, were cultured for 5 days in the presenceof Aβ1-40 at a concentration of 5 μM for stimulation. As a positivecontrol, additional cells were cultured with the T cell mitogen, PHA,and, as a negative control, cells were cultured without added peptide.

Splenocytes from aged PDAPP mice passively immunized with variousanti-Aβ antibodies were stimulated in vitro with AN1792 andproliferative and cytokine responses were measured. The purpose of theseassays was to determine if passive immunization facilitated antigenpresentation, and thus priming of T cell responses specific for AN1792.No AN1792-specific proliferative or cytokine responses were observed inmice passively immunized with the anti-Aβ antibodies.

Example II Therapeutic Efficacy of Anti-Aβ Antibodies: mAb 2H3, mAb10D5, mAb 266, mAb 21F12, mAb 3D6, mAb 16C11 and pAb Aβ1-42

In a second study, treatment with 10D5 was repeated and two additionalanti-Aβ antibodies were tested, monoclonals 3D6 (Aβ1-5) and 16C11(Aβ33-42). Control groups received either PBS or an irrelevantisotype-matched antibody (TM2a). The mice were older (11.5-12 month oldheterozygotes) than in the previous study, otherwise the experimentaldesign was the same. Once again, after six months of treatment, 10D5reduced plaque burden by greater than 80% relative to either the PBS orisotype-matched antibody controls (p=0.003). One of the other antibodiesagainst Aβ, 3D6, was equally effective, producing an 86% reduction(p=0.003). In contrast, the third antibody against the peptide, 16C11,failed to have any effect on plaque burden. Similar findings wereobtained with Aβ42 ELISA measurements.

These results demonstrate that an antibody response against Aβ peptide,in the absence of T cell immunity, is sufficient to decrease amyloiddeposition in PDAPP mice, but that not all anti-Aβ antibodies areequally efficacious. Antibodies directed to epitopes comprising aminoacids 1-5 or 3-7 of Aβ are particularly efficacious. In summary, it canbe demonstrated that passively administered antibodies against Aβ (i.e.,passive immunization) reduces the extent of plaque deposition in a mousemodel of Alzheimer's disease.

Example III Monitoring of Antibody Binding in the CNS

This Example demonstrates that when held at modest serum concentrations(25-70 μg/ml), the antibodies gained access to the CNS at levelssufficient to decorate β-amyloid plaques.

To determine whether antibodies against Aβ could be acting directlywithin the CNS, brains taken from saline-perfused mice at the end of theExample II, were examined for the presence of theperipherally-administered antibodies. Unfixed cryostat brain sectionswere exposed to a fluorescent reagent against mouse immunoglobulin (goatanti-mouse IgG-Cy3). Plaques within brains of the 10D5 and 3D6 groupswere strongly decorated with antibody, while there was no staining inthe 16C11 group. To reveal the full extent of plaque deposition, serialsections of each brain were first immunoreacted with an anti-Aβantibody, and then with the secondary reagent. 10D5 and 3D6, followingperipheral administration, gained access to most plaques within the CNS.The plaque burden was greatly reduced in these treatment groups comparedto the 16C11 group. Antibody entry into the CNS was not due to abnormalleakage of the blood-brain barrier since there was no increase invascular permeability as measured by Evans Blue in PDAPP mice. Inaddition, the concentration of antibody in the brain parenchyma of agedPDAPP mice was the same as in non-transgenic mice, representing 0.1% ofthe antibody concentration in serum (regardless of isotype).

These data indicate that peripherally administered antibodies can enterthe CNS where they can directly trigger amyloid clearance. It is likelythat 16C11 also had access to the plaques but was unable to bind.

Example IV Ex Vivo Screening Assay for Activity of an Antibody AgainstAmyloid Deposits

To examine the effect of antibodies on plaque clearance, we establishedan ex vivo assay in which primary microglial cells were cultured withunfixed cryostat sections of either PDAPP mouse or human AD brains.Microglial cells were obtained from the cerebral cortices of neonateDBA/2N mice (1-3 days). The cortices were mechanically dissociated inHBSS⁻ (Hanks' Balanced Salt Solution, Sigma) with 50 μg/ml DNase I(Sigma). The dissociated cells were filtered with a 100 μm cell strainer(Falcon), and centrifuged at 1000 rpm for 5 minutes. The pellet wasresuspended in growth medium (high glucose DMEM, 10% FBS, 25 ng/mlrmGM-CSF), and the cells were plated at a density of 2 brains per T-75plastic culture flask. After 7-9 days, the flasks were rotated on anorbital shaker at 200 rpm for 2 h at 37° C. The cell suspension wascentrifuged at 1000 rpm and resuspended in the assay medium.

10-μm cryostat sections of PDAPP mouse or human AD brains (post-morteminterval <3 hr) were thaw mounted onto poly-lysine coated round glasscoverslips and placed in wells of 24-well tissue culture plates. Thecoverslips were washed twice with assay medium consisting of H—SFM(Hybridoma-serum free medium, Gibco BRL) with 1% FBS, glutamine,penicillin/streptomycin, and 5 ng/ml rmGM-CSF (R&D). Control or anti-Aβantibodies were added at a 2× concentration (5 μg/ml final) for 1 hour.The microglial cells were then seeded at a density of 0.8×10⁶ cells/mlassay medium. The cultures were maintained in a humidified incubator(37° C., 5% CO₂) for 24 hr or more. At the end of the incubation, thecultures were fixed with 4% paraformaldehyde and permeabilized with 0.1%Triton-X100. The sections were stained with biotinylated 3D6 followed bya streptavidin/Cy3 conjugate (Jackson ImmunoResearch). The exogenousmicroglial cells were visualized by a nuclear stain (DAPI). The cultureswere observed with an inverted fluorescent microscope (Nikon, TE300) andphotomicrographs were taken with a SPOT digital camera using SPOTsoftware (Diagnostic instruments). For Western blot analysis, thecultures were extracted in 8M urea, diluted 1:1 in reducing tricinesample buffer and loaded onto a 16% tricine gel (Novex). After transferonto immobilon, blots were exposed to 5 μg/ml of the pabAβ42 followed byan HRP-conjugated anti-mouse antibody, and developed with ECL (Amersham)

When the assay was performed with PDAPP brain sections in the presenceof 16C11 (one of the antibodies against Aβ that was not efficacious invivo), β-amyloid plaques remained intact and no phagocytosis wasobserved. In contrast, when adjacent sections were cultured in thepresence of 10D5, the amyloid deposits were largely gone and themicroglial cells showed numerous phagocytic vesicles containing Aβ.Identical results were obtained with AD brain sections; 10D5 inducedphagocytosis of AD plaques, while 16C11 was ineffective. In addition,the assay provided comparable results when performed with either mouseor human microglial cells, and with mouse, rabbit, or primate antibodiesagainst Aβ.

Table 7 compares Aβ binding versus phagocytosis for several differentantibody binding specificities. It can be seen that antibodies bindingto epitopes within aa 1-7 both bind and clear amyloid deposits, whereasantibodies binding to epitopes within amino acids 4-10 bind withoutclearing amyloid deposits. Antibodies binding to epitopes C-terminal toresidue 10 neither bind nor clear amyloid deposits. TABLE 7 Analysis ofEpitope Specificity Antibody epitope isotype Staining PhagocytosisN-Term mab 3D6 1-5 IgG2b + + 10D5 3-7 IgG1 + + 22C8 3-7 IgG2a + + 6E10 5-10 IgG1 + − 14A8  4-10 rat IgG1 + − aa 13-28 18G11 10-18 rat IgG1 − −266 16-24 IgG1 − − 22D12 18-21 IgG2b − − C-Term 2G3 −40 IgG1 − − 16C11−40/−42 IgG1 − − 21F12 −42 IgG2a − − Immune serum rabbit (CFA) 1-6 + +mouse (CFA) 3-7 + + mouse (QS-21) 3-7 + + monkey (QS-21) 1-5 + + mouse(MAP1-7) + +

Table 8 shows results obtained with several antibodies against Aβ,comparing their abilities to induce phagocytosis in the ex vivo assayand to reduce in vivo plaque burden in passive transfer studies.Although 16C11 and 21F12 bound to aggregated synthetic Aβ peptide withhigh avidity, these antibodies were unable to react with β-amyloidplaques in unfixed brain sections, could not trigger phagocytosis in theex vivo assay, and were not efficacious in vivo. 10D5, 3D6, and thepolyclonal antibody against Aβ were active by all three measures. Theseresults show that efficacy in vivo is due to direct antibody mediatedclearance of the plaques within the CNS, and that the ex vivo assay ispredictive of in vivo efficacy. TABLE 8 The ex vivo assay as predictorof in vivo efficacy Avidity for Binding to aggregated β-amyloid Ex vivoIn Antibody Isotype Aβ (pM) plaques efficacy vivo efficacy monoclonal3D6 IgG2b 470 + + + 10D5 IgG1  43 + + + 16C11 IgG1  90 − − − 21F12 IgG2a500 − − − TM2a IgG1 — − − − polyclonal 1-42 mix 600 + + +

The same assay has been used to test clearing activity of an antibodyagainst a fragment of synuclein referred to as NAC. Synuclein has beenshown to be an amyloid plaque-associated protein. An antibody to NAC wascontacted with a brain tissue sample containing amyloid plaques, andmicroglial cells, as before. Rabbit serum was used as a control.Subsequent monitoring showed a marked reduction in the number and sizeof plaques indicative of clearing activity of the antibody.

Confocal microscopy was used to confirm that Aβ was internalized duringthe course of the ex vivo assay. In the presence of control antibodies,the exogenous microglial cells remained in a confocal plane above thetissue, there were no phagocytic vesicles containing Aβ, and the plaquesremained intact within the section. In the presence of 10D5, nearly allplaque material was contained in vesicles within the exogenousmicroglial cells. To determine the fate of the internalized peptide,10D5 treated cultures were extracted with 8M urea at varioustime-points, and examined by Western blot analysis. At the one hour timepoint, when no phagocytosis had yet occurred, reaction with a polyclonalantibody against Aβ revealed a strong 4 kD band (corresponding to the Aβpeptide). Aβ immunoreactivity decreased at day 1 and was absent by day3. Thus, antibody-mediated phagocytosis of Aβ leads to its degradation.

To determine if phagocytosis in the ex vivo assay was Fc-mediated,F(ab′)2 fragments of the anti-Aβ antibody 3D6 were prepared. Althoughthe F(ab′)2 fragments retained their full ability to react with plaques,they were unable to trigger phagocytosis by microglial cells. Inaddition, phagocytosis with the whole antibody could be blocked by areagent against murine Fc receptors (anti-CD16/32). These data indicatethat in vivo clearance of Aβ occurs through Fc-receptor mediatedphagocytosis.

Example V Passage of Antibodies Through the Blood-Brain Barrier

This example determines the concentration of antibody delivered to thebrain following intravenous injection into a peripheral tissue of eithernormal or PDAPP mice. Following treatment, PDAPP or control normal micewere perfused with 0.9% NaCl. Brain regions (hippocampus or cortex) weredissected and rapidly frozen. Brain were homogenized in 0.1%triton+protease inhibitors. Immunoglobulin was detected in the extractsby ELISA. F(ab)′2 goat anti-mouse IgG were coated onto an RIA plate ascapture reagent. The serum or the brain extracts were incubated for 1hr. The isotypes-were detected with anti-mouse IgG1-HRP or IgG2a-HRP orIgG2b-HRP (Caltag). Antibodies, regardless of isotype, were present inthe CNS at a concentration that is 1:1000 that found in the blood. Forexample, when the concentration of IgG1 was three times that of IgG2a inthe blood, it was three times IgG2a in the brain as well, both beingpresent at 0.1% of their respective levels in the blood. This result wasobserved in both transgenic and nontransgenic mice indicating that thePDAPP does not have a uniquely leak blood brain barrier.

Example VI Cloning and Sequencing of the Mouse 3D6 Variable Regions

Cloning and Sequence Analysis of 3D6 VH. The heavy chain variable VHregion of 3D6 was cloned by RT-PCR using mRNA prepared from hybridomacells by two independent methods. In the first, consensus primers wereemployed to VH region leader peptide encompassing the translationinitiation codon as the 5′ primer (DNA #3818-3829), and a g2b (DNA#3832) constant regions specific 3′ primer. The sequences from PCRamplified product, as well as from multiple, independently-derivedclones, were in complete agreement with one another. As a further checkon the sequence of the 3D6 VH region, the result was confirmed bysequencing a VH fragment obtained by 5′ RACE RT-PCR methodology and the3′ g2b specific primer (DNA #3832). Again, the sequence was derived fromthe PCR product, as well as multiple, independently-isolated clones.Both sequences are in complete agreement with one another, (with theexception of V8I substitution in the leader region from the 5′ RACEproduct), indicating that the sequences are derived from the mRNAencoding the VH region of 3D6. The nucleotide (SEQ ID NO:3) and aminoacid sequence (SEQ ID NO:4) of the VH region of 3D6 are set forth inTable 9A and in FIG. 2, respectively. TABLE 9A Mouse 3D6 VH NucleotideSequence ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGCGTCTCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACTATGGCATGTCTTGGGTTCGCCAGAATTCAGACAAGAGGCTGGAGTGGGTTGCATCCATTAGGAGTGGTGGTGGTAGAACCTACTATTCAGACAATGTAAAGGGCCGATTCACCATCTCCAGAGAGAATGCCAAGAACACCCTGTACCTGCAAATGAGTAGTCTGAAGTCTGAGGACACGGCCTTGTATTATTGTGTCAGATATGATCACTATAGTGGTAGCTCCGACTACTGGGGCCAGGGCACCACT (SEQ ID NO: 3)*Leader peptide is underlined.

Cloning and Sequence Analysis of 3D6 VL. The light chain variable VLregion of 3D6 was cloned in an analogous manner as the VH region. In thefirst trial, a consensus primer set was designed for amplification ofmurine VL regions as follows: 5′ primers (DNA #3806-3816) were designedto hybridize to the VL region encompassing the translation initiationcodon, and a 3′ primer (DNA#3817) was specific for the murine Ck regiondownstream of the V-J joining region. DNA sequence analysis of the PCRfragment, as well as independently-derived clones isolated using thisconsensus light chain primer set, revealed that the cDNA obtained wasderived from a non-functionally rearranged message as the sequencecontained a frameshift mutation between the V-J region junction.

In a second trial, 5′RACE was employed to clone a second VL encodingcDNA. DNA sequence analysis of this product (consensus 11) showed itencoded a functional mRNA. Thus, it can be concluded that the sequenceencodes the correct 3D6 light chain mRNA. The nucleotide (SEQ ID NO:1)and amino acid sequence (SEQ ID. NO:2) of the VL region of 3D6 are setforth in Table 9B and in FIG. 1, respectively. TABLE 9B Mouse 3D6 VLNucleotide Sequence ATGATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAACGGTTATGTTGTGATGACCCAGACTCCACTCACTTTGTCGCTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTTACACTGAAAATCAGCAGAATAGAGGCTGAGGATTTGGGACTTTATTATTGCTGGCAAGGTACACATTTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 1)* Leader peptide is underlined

Primers used for the cloning of the 3D6 VL cDNA are set forth in TABLE10 Coding DNA Size Strand? DNA Sequence Comments 3806 40 YesACT.AGT.CGA.CAT.GAA.GTT.GCC.TGT.TA mouse kappa variableG.GCT.GTT.GGT.GCT.G primer 1 (SEQ ID NO:39) MKV PRIMER 1, MRC set; % A+ T = 50.00 [20]; % C + G = 50.00 [20] Davis, Botstein, Roth MeltingTemp C. 72.90 3807 39 Yes ACT.AGT.CGA.CAT.GGA.GWC.AGA.CAC.AC mouse kappavariable T.CCT.GYT.ATG.GGT primer 2 (SEQ ID NO:40) MKV PRIMER 2, MRC set% A + T = 46.15 [18]; % C + G = 48.72 [19] Davis, Botstein, Roth MeltingTemp C. 72.05 3808 40 Yes ACT.AGT.CGA.CAT.GAG.TGT.GCT.CAC.TC mouse kappavariable A.GGT.CCT.GGS.GTT.G primer 3 (SEQ ID N0:41) MKV PRIMER 3, MRCset; % A + T = 45.00 [18]; % C + G = 52.50 [21] Davis, Botstein, RothMelting Temp C. 73.93 3809 43 Yes ACT.AGT.CGA.CAT.GAG.GRC.CCC.TGC.TCmouse kappa variable A.GWT.TYT.TGG.MWT.CTT.G primer 4 (SEQ ID NO:42) MKVPRIMER 4, MRC set; % A + T = 41.86 [18]; % C + G = 46.51 [20] Davis,Botstein, Roth Melting Temp C. 72.34 3810 40 YesACT.AGT.CGA.CAT.GGA.TTT.WCA.GGT.GC mouse kappa variableA.GAT.TWT.CAG.CTT.C primer 5 (SEQ ID NO:43) MKV PRIMER 5, MRC set % A+ T = 52.50 [21]; % C + G = 42.50 [17] Davis, Botstein, Roth MeltingTemp C. 69.83 3811 37 Yes ACT.AGT.CGA.CAT.GAG.GTK.CYY.TGY.TS mouse kappavariable A.GYT.YCT.GRG.G primer 6 (SEQ ID NO:44) MKV PRIMER 6, MRC set;% A + T = 37.84 [14 , C + G = 40.54 [15] Davis, Botstein, Roth MeltingTemp C. 68.01 3812 41 Yes ACT.AGT.CGA.CAT.GGG.CWT.CAA.GAT.GG mouse kappavariable A.GTC.ACA.KWY.YCW.GG primer 7 (SEQ ID NO:45) MKV PRIMER 7, MRCset; % A + T = 39.02 [16]; % C + G = 46.34 [19] Davis, Botstein, RothMelting Temp C. 71.70 3813 41 Yes ACT.AGT.CGA.CAT.GTG.GGG.AYC.TKT.TTmouse kappa variable Y.CMM.TTT.TTC.AAT.TG primer 8 (SEQ ID NO:46) MKVPRIMER 8, MRC set; % A + T = 53.66 [22]; % C = G = 34.15 [14] Davis,Botstein, Roth Melting Temp C. 66.70 3814 35 YesACT.AGT.CGA.CAT.GGT.RTC.CWC.ASC.TC mouse kappa variable A.GTT.CCT.TGprimer 9 (SEQ ID NO:47) MKV PRIMER 9, MRC set. % A + T = 45.71 [16]; % C+ G = 45.71 [16] Davis, Botstein, Roth Melting Temp C. 69.36 3815 37 YesACT.AGT.CGA.CAT.GTA.TAT.ATG.TTT.GT mouse kappa variable T.GTC.TAT.TTC.Tprimer 10 (SEQ ID NO:48) MKV PRIMER 10, MRC set; % A + T = 70.27 [26]; %C + G = 29.73 [11] Davis, Botstein, Roth Melting Temp C. 63.58 3816 38Yes ACT.AGT.CGA.CAT.GGA.AGC.CCC.AGC.TC mouse kappa variableA.GCT.TCT.CTT.CC primer 11 (SEQ ID NO:49) MKV PRIMER 11, MRC set; % A+ T = 44.74 [17]; % C + G = 55.26 [21] Davis, Botstein, Roth MeltingTemp C. 74.40 3817 27 No GGA.TCC.CGG.GTG.GAT.GGT.GGG.AAG.AT mouse kappalight chain G reverse primer, aa 116-122; (SEQ ID NO:50) Ck constantregion primer, MRC set + SmaI site; % A + T = 47.06 [8]; % C + G = 52.94[9] Davis,Botstein, Roth Melting Temp C. 57.19 3818 37 YesACT.AGT.CGA.CAT.GAA.ATG.CAG.CTG.GG mouse heavy variable T.CAT.STT.CTT.Cprimer 1 (SEQ ID NO:51) MHV primer 1, MRC set; 3819 36 YesACT.AGT.CGA.CAT.GGG.ATG.GAG.CTR.TA mouse heavy variable T.CAT.SYT.CTTprimer 2 (SEQ ID NO:52) MHV primer 2, MRC set; 3820 37 YesACT.AGT.CGA.CAT.GAA.GWT.GTG.GTT.AA mouse heavy variable T.CTG.GGT.TTT.Tprimer 3 (SEQ ID NO:53) MHV primer 3, MRC set; 3821 35 YesACT.AGT.CGA.CAT.GRA.CTT.TGG.GYT.CA mouse heavy variable G.CTT.GRT.TTprimer 4 (SEQ ID NO:54) MHV primer 4, MRC set; 3822 40 YesACT.AGT.CGA.CAT.GGA.CTC.CAG.GCT.CA mouse heavy variableA.TTT.AGT.TTT.CCT.T primer 5 (SEQ ID NO:55) MHV primer 5, MRC set; 382337 Yes ACT.AGT.CGA.CAT.GGC.TGT.CYT.RGS.GC mouse heavy variableT.RCT.CTT.CTG.C primer 6 (SEQ ID NO:56) MHV primer 6, MRC set; 3824 36Yes ACT.AGT.CGA.CAT.GGR.ATG.GAG.CKG.GR mouse heavy variableT.CTT.TMT.CTT primer 7 (SEQ ID NO:57) MHV primer 7, MRC set; 3825 33 YesACT.AGT.CGA.CAT.GAG.AGT.GCT.GAT.TC mouse heavy variable T.TTT.GTG primer8 (SEQ ID NO:58) MHV primer 8, MRC set; 3826 40 YesACT.AGT.CGA.CAT.GGM.TTG.GGT.GTG.GA mouse heavy variableM.CTT.GCT.ATT.CCT.G primer 9 (SEQ ID NO:59) MHV primer 9, MRC set; 382737 Yes ACT.AGT.CGA.CAT.GGG.CAG.ACT.TAC.AT mouse heavy variableT.CTC.ATT.CCT.G primer 10 (SEQ ID NO:60) MHV primer 10, MRC set; 3828 38Yes ACT.AGT.CGA.CAT.GGA.TTT.TGG.GCT.GA mouse heavy variableT.TTT.TTT.TAT.TG primer 11 (SEQ ID NO:61) MHV primer 11, MRC set; 382937 Yes ACT.AGT.CGA.CAT.GAT.GGT.GTT.AAG.TC mouse heavy variableT.TCT.GTA.CCT.G primer 12 (SEQ ID NO:62) MHV primer 12, MRC set; 3832 27No GGA.TCC.CGG.GAG.TGG.ATA.GAC.tGA.TG mouse IgG2h heavy chain G reverseprimer (SEQ ID NO:63) aa position 119-124, MRC set;

From N-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of aminoacids to each domain is in accordance with the numbering convention ofKabat et al., supra.

Expression of Chimeric 3D6 Antibody: The variable heavy and light chainregions were re-engineered to encode splice donor sequences downstreamof the respective VDJ or VJ junctions, and cloned into the mammalianexpression vector pCMV-hγ1 for the heavy chain, and pCMV-hκ1 for thelight chain. These vectors encode human γ1 and Ck constant regions asexonic fragments downstream of the inserted variable region cassette.Following sequence verification, the heavy chain and light chainexpression vectors were co-transfected into COS cells. Two differentheavy chain clones (H2.2 & H3.2) were independently co-transfected with3 different chimeric light chain clones (L3, L4, &L10) to confirmreproducibility of the result. A chimeric 21.6 antibody transfection wascarried out as a positive control for the vectors. Conditioned media wascollected 48 hrs post transfection and assayed by western blot analysisfor antibody production or ELISA for Aβ binding.

The multiple transfectants all expressed heavy chain+light chaincombinations which are recognized by a goat anti-human IgG (H+L)antibody on a western blot.

Direct binding of 3D6 and chimeric 3D6 (PK1614) antibodies to Aβ wastested by ELISA analysis. Chimeric 3D6 was found to bind to Aβ with highavidity, similar to that demonstrated by 3D6 (FIG. 3A). Furthermore, anELISA based competitive inhibition assay revealed that the chimeric 3D6and the murine 3D6 antibody competed equally with biotinylated-3D6binding to Aβ (FIG. 3B). The chimeric antibody displayed bindingproperties indistinguishable from the 3D6 reference sample. TABLE 11Conc (μg/ml) 3D6 PK1614 IgG1 0.037 119.3 0.11 118.6 118.9 0.33 99.771.25 1 98.63 84.53 134.4

Moreover, both 3D6 and PK1614 were effective at clearing Aβ plaques. Theex vivo assay demonstrates that as the concentration of antibodyincreases, the amount of Aβ decreases in a similar manner for bothmurine and chimeric 3D6 antibodies. Hence, it can be concluded that thesequences encode functional 3D6 heavy chain and light chainsrespectively.

Example VII 3D6 Humanization

Homology/Molecular Modeling. In order to identify key structuralframework residues in the murine 3D6 antibody, a three-dimensional modelwas generated based on the closest murine antibodies for the heavy andlight chains. For this purpose, an antibody designated 1CR9 was chosenas a template for modeling the 3D6 light chain (PDB ID: 1CR9, Kanyo etal., supra), and an antibody designated 1OPG was chosen as the templatefor modeling the heavy chain. (PDB ID: 1OPG Kodandapani et al., supra).(See also Table 1.) Amino acid sequence alignment of 3D6 with the lightchain and heavy chain of these antibodies revealed that, with theexception of CDR3 of the heavy chain, the 1CR9 and 1OPG antibodies sharesignificant sequence homology with 3D6. In addition, the CDR loops ofthe selected antibodies fall into the same canonical Chothia structuralclasses as do the CDR loops of 3D6, again excepting CDR3 of the heavychain. Therefore, 1CR9 and 1OPG were initially selected as antibodies ofsolved structure for homology modeling of 3D6.

A first pass homology model of 3D6 variable region based on theantibodies noted above was constructed using the Look & SegMod ModulesGeneMine (v3.5) software package. This software was purchased under aperpetual license from Molecular Applications Group (Palo Alto, Calif.).This software package, authored by Drs. Michael Levitt and Chris Lee,facilitates the process of molecular modeling by automating the stepsinvolved in structural modeling a primary sequence on a template ofknown structure based on sequence homology. Working on a SiliconGraphics IRIS workstation under a UNIX environment, the modeledstructure is automatically refined by a series of energy minimizationsteps to relieve unfavorable atomic contacts and optimize electrostaticand van der Walls interactions.

A further refined model was built using the modeling capability ofQuanta®. A query of the PDB database with CDR3 of the heavy chain of 3D6identified 1qkz as most homologous and having the identical number ofresidues as 3D6. Hence, CDR3 of the heavy chain of 3D6 was modeled usingthe crystal structure of 1qkz as template. The α-carbon backbone traceof the 3D6 model is shown in FIG. 4. The VH domain is shown as astippled line, and VL domain is shown as a solid line, and CDR loops areindicated in ribbon form.

Selection of Human Acceptor Antibody Sequences. Suitable human acceptorantibody sequences were identified by computer comparisons of the aminoacid sequences of the mouse variable regions with the sequences of knownhuman antibodies. The comparison was performed separately for the 3D6heavy and light chains. In particular, variable domains from humanantibodies whose framework sequences exhibited a high degree of sequenceidentity with the murine VL and VH framework regions were identified byquery of the Kabat Database using NCBI BLAST (publicly accessiblethrough the National Institutes of Health NCBI internet server) with therespective murine framework sequences.

Two candidate sequences were chosen as acceptor sequences based on thefollowing criteria: (1) homology with the subject sequence; (2) sharingcanonical CDR structures with the donor sequence; and (3) not containingany rare amino acid residues in the framework regions. The selectedacceptor sequence for VL is Kabat ID Number (KABID) 019230 (GenbankAccession No. S40342), and for VH is KABID 045919 (Genbank Accession No.AF115110). First versions of humanized 3D6 antibody utilize theseselected acceptor antibody sequences.

Substitution of Amino Acid Residues. As noted supra, the humanizedantibodies of the invention comprise variable framework regionssubstantially from a human immunoglobulin (acceptor immunoglobulin) andcomplementarity determining regions substantially from a mouseimmunoglobulin (donor immunoglobulin) termed 3D6. Having identified thecomplementarity determining regions of 3D6 and appropriate humanacceptor immunoglobulins, the next step was to determine which, if any,residues from these components to substitute to optimize the propertiesof the resulting humanized antibody. The criteria described supra wereused to select residues for substitution.

FIGS. 1 and 2 depict alignments of the original murine 3D6 VL and VH,respectively, with the respective version 1 of the humanized sequence,the corresponding human framework acceptor sequence and, lastly, thehuman germline V region sequence showing highest homology to the humanframework acceptor sequence. The shaded residues indicate the canonical(solid fill), vernier (dotted outline), packing (bold), and rare aminoacids (bold italics), and are indicated on the figure. The asterisksindicate residues backmutated to murine residues in the human acceptorframework sequence, and CDR regions are shown overlined. A summary ofthe changes incorporated into version 1 of humanized 3D6 VH and VL ispresented in Table 12. TABLE 12 Summary of changes in humanized 3D6.v1Changes VL (112 residues) VH (119 residues) Hu->Mu: Framework  4/1123/119 (1 canon, 1 packing) CDR1  6/16 3/5  CDR2 4/7 7/14 CDR3 5/8 4/10Hu->Mu 19/112 (17%) 17/119 (14%) Mu->Hu: Framework  13/112 14/119Backmutation notes 1. I2V which is a canonical 4. S49A Vernier/beneaththe po sition. CDRs. 2. Y36L which is a packing 5. A93V which is apacking residue and also lies under the and vernier zone residue CDRs 6.K94R which is a canonical 3. L46R which is a packing residue residue andlies beneath the CDRs Acceptor notes 7. KABID 019230/Genbank 11.KABID045919/Genbank Acc#S40342 Acc#AF115110 8. Hu κ LC subgroup II 12.Hu HC subgroup III 9. CDRs from same canonical 13. CDRs from samecanonical structural group as donor structural group as donor (m3D6)(m3D6) L1 = class 4 H1 = class 1 L2 = class 1 H2 = class3 L3 = class114. Recognizes capsular 10. Unknown specificity polysaccharide ofNeisseria meningitidis Acceptor Germline 15. VH3-23 16. A3 & A19

Tables 13 and 14 set forth Kabat numbering keys for the various lightand heavy chains, respectively. TABLE 13 Key to Kabat Numbering forLight Chain A19- mouse HUM KABID Germ- 3D6VL 3D6VL 019230 line (aa 1-112(aa 1-112 (aa 1-112 (SEQ SEQ ID SEQ ID SEQ ID ID KAB # # TYPE NO: 2) NO:5) NO: 6) NO: 7) Comment  1 1 FR1 Y Y D D Rare mouse, may contact CDR  22 V V I I Canonical/CDR contact  3 3 V V V V  4 4 M M M M  5 5 T T T T 6 6 Q Q Q Q  7 7 T S S S  8 8 P P P P  9 9 L L L L 10 10 T S S S 11 11L L L L 12 12 S P P P 13 13 V V V V 14 14 T T T T 15 15 I P P P 16 16 GG G G 17 17 Q E E E 18 18 P P P P 19 19 A A A A 20 20 S S S S 21 21 I II I 22 22 S S S S 23 23 C C C C 24 24 CDR1 K K R R 25 25 S S S S 26 26 SS S S 27 27 Q Q Q Q   27A 28 S S S S   27B 29 L L L L   27C 30 L L L L  27D 31 D D H H   27E 32 S S S S 28 33 D D N N 29 34 G G G G 30 35 K KY Y 31 36 T T N N 32 37 Y Y Y Y 33 38 L L L L 34 39 N N D D 35 40 FR2 WW W W 36 41 L L Y Y Packing residue 37 42 L L L L 38 43 Q Q Q Q 39 44 RK K K 40 45 P P P P 41 46 G G G G 42 47 Q Q Q Q 43 48 S S S S 44 49 P PP P 45 50 K Q Q Q 46 51 R R L L Packing residue 47 52 L L L L 48 53 I II I 49 54 Y Y Y Y 50 55 CDR2 L L L L 51 56 V V G G 52 57 S S S S 53 58 KK N N 54 59 L L R R 55 60 D D A A 56 61 S S S S 57 62 FR3 G G G G 58 63V V V V 59 64 P P P P 60 65 D D D D 61 66 R R R R 62 67 F F F F 63 68 TS S S 64 69 G G G G 65 70 S S S S 66 71 G G G G 67 72 S S S S 68 73 G GG G 69 74 T T T T 70 75 D D D D 71 76 F F F F 72 77 T T T T 73 78 L L LL 74 79 K K K K 75 80 I I I I 76 81 S S S S 77 82 R R R R 78 83 I V V V79 84 E E E E 80 85 A A A A 81 86 E E E E 82 87 D D D D 83 88 L V V V 8489 G G G G 85 90 L V V V 86 91 Y Y Y Y 87 92 Y Y Y Y 88 93 C C C C 89 94CDR3 W W M M 90 95 Q Q Q Q 91 96 G G A A 92 97 T T L L 93 98 H H Q Q 9499 F F T T 95 100 P P P P 96 101 R R R 97 102 T T T 98 103 FR4 F F F 99104 G G G 100  105 G Q Q 101  106 G G G 102  107 T T T 103  108 K K K104  109 L V V 105  110 E E E 106  111 I I I  106A 112 K K K

TABLE 14 Key to Kabat Numbering for Heavy Chain VH3-23 Mouse HUM KABIDGerm- 3D6 VH 3D6 VH 045919 line (aa 1-119 (aa 1-119 (SEQ (SEQ SEQ ID SEQID ID ID KAB # # TYPE NO: 4) NO: 8) NO: 9) NO: 10) Comment  1 1 FR1 E EE E  2 2 V V V V  3 3 K Q Q Q  4 4 L L L L  5 5 V L L L  6 6 E E E E  77 S S S S  8 8 G G G G  9 9 G G G G 10 10 G G G G 11 11 L L L L 12 12 VV V V 13 13 K Q Q Q 14 14 P P P P 15 15 G G G G 16 16 A G G G 17 17 S SS S 18 18 L L L L 19 19 K R R R 20 20 L L L L 21 21 S S S S 22 22 C C CC 23 23 A A A A 24 24 A A A A 25 25 S S S S 26 26 G G G G 27 27 F F F F28 28 T T T T 29 29 F F F F 30 30 S S S S 31 31 CDR1 N N S S 32 32 Y Y YY 33 33 G G A A 34 34 M M V M 35 35 S S S S 36 36 FR2 W W W W 37 37 V VV V 38 38 R R R R 39 39 Q Q Q Q 40 40 N A A A Rare mouse, replace w/Hum41 41 S P P P 42 42 D G G G Rare mouse, replace w/Hum 43 43 K K K K 4444 R G G G 45 45 L L L L 46 46 E E E E 47 47 W W W W 48 48 V V V V 49 49A A S S CDR contact/veneer 50 50 CDR2 S S A A 51 51 I I I I 52 52 R R SS   52A 53 S S G G 53 54 G G S S 54 55 G G G G 55 56 G G G G 56 57 R R SS 57 58 T T T T 58 59 Y Y Y Y 59 60 Y Y Y Y 60 61 S S A A 61 62 D D D D62 63 N N S S 63 64 V V V V 64 65 K K K K 65 66 G G G G 66 67 FR3 R R RR 67 68 F F F F 68 69 T T T T 69 70 I I I I 70 71 S S S S 71 72 R R R R72 73 E D D D 73 74 N N N N 74 75 A A A S 75 76 K K K K 76 77 N N N N 7778 T S S T 78 79 L L L L 79 80 Y Y Y Y 80 81 L L L L 81 82 Q Q Q Q 82 83M M M M   82A 84 S N N N   82B 85 S S S S   82C 86 L L L L 83 87 K R R R84 88 S A A A 85 89 E E E E 86 90 D D D D 87 91 T T T T 88 92 A A A A 8993 L L L V 90 94 Y Y Y Y 91 95 Y Y Y Y 92 96 C C C C 93 97 V V A APacking residue, use mouse 94 98 R R K K Canonical, use mouse 95 99 CDR3Y Y D 96 100 D D N 97 101 H H Y 98 102 Y Y D 99 103 S S F 100  104 G G W 100A 105 S S S  100B 106 S S G  100C 107 — — T  100D 108 — — F 101  109D D D 102  110 Y Y Y 103  111 FR4 W W W 104  112 G G G 105  113 Q Q Q106  114 G G G 107  115 T T T 108  116 T L L 109  117 V V V 110  118 T TT 111  119 V V V 112  120 S S S 113  121 S S S

The humanized antibodies preferably exhibit a specific binding affinityfor Aβ of at least 10⁷, 10⁸, 10⁹ or 10¹⁰ M⁻¹. Usually the upper limit ofbinding affinity of the humanized antibodies for Aβ is within a factorof three, four or five of that of 3D6 (i.e., ˜10⁹ M⁻¹). Often the lowerlimit of binding affinity is also within a factor of three, four or fiveof that of 3D6.

Assembly and Expression of Humanized 3D6 VH and VL, Version 1 Briefly,for each V region, 4 large single stranded overlapping oligonucleotideswere synthesized. In addition, 4 short PCR primers were synthesized foreach V region to further facilitate assembly of the particular V region.The DNA sequences of the oligonucleotides employed for this purpose areshown in Table 15. TABLE 15 DNA oligonucleotides DNA# SIZE Coding?Sequence comments 4060 136 Yes tccgc aagct tgccg ccacc hum 3D6 VL-AATGGA CATGC GCGTG CCCGC CCAGC TGCTG GGCCT GCTGA TGCTG TGGGT GTCCG GCTCCTCCGG CTACG TGGTG ATGAC CCAGT CCCCC CTGTC CCTGC CCGTG ACCCC CGGCG A (SEQID NO:17) 4061 131 No CTGGG GGGAC TGGCC GGGCT hum 3D6 VL-B TCTGC AGCAGCCAGT TCAGG TAGGT CTTGC CGTCG GAGTC CAGGA GGGAC TGGGA GGACT TGCAG GAGATGGAGG CGGGC TCGCC GGGGG TCACG GGCAG GGACA GGGGG G (SEQ. ID NO:18) 4062146 Yes ACCTG AACTG GCTGC TGCAG hum 3D6 VL-C AAGCC CGGCC AGTCC CCCCAGCGCC TGATC TACCT GGTGT CCAAG CTGGA CTCCG GCGTG CCCGA CCGCT TCTCC GGCTCCGGCT CCGGC ACCGA CTTCA CCCTG AAGAT CTCCC GCGTG GAGGC C (SEQ ID NO:19)4063 142 No aattc tagga tccac tcacg hum 3D6VL-D CTTGA TCTCC ACCTT GGTGCCCTGG CCGAA GGTGC GGGGG AAGTG GGTGC CCTGC CAGCA GTAGT ACACG CCCAC GTCCTCGGCC TCCAC GCGGG AGATC TTCAG GGTGA AGTCG GTGCC GG (SEQ ID NO:20) 406416 No CTGGG GGGAC TGGCC G hum 3D6 VL A + B (SEQ ID NO: 21) back % A + T= 18.75 [3]; % C + G = 81.2 [13] Davis, Botstein, Roth Melting Temp C.66.96 4065 22 Yes ACCTG AACTG GCTGC TGCAG hum 3D6 VL C + D AA forward(SEQ ID NO.22) % A + T = 45.45 [10]; % C + G = 54.55 [12] Davis,Botstein, Roth Melting Temp C. 64.54 4066 138 Yes acaga aagct tgccgccacc hum 3D6 VH-A ATGGA GTTTG GGCTG AGCTG GCTTT TTCTT GTGGC TATTT TAAAAGGTGT CCAGT GTGAG GTGCA GCTGC TGGAG TCCGG CGGCG GCCTG GTGCA GCCCG GCGGCTCCCT GCGCC TGT (SEQ ID NO:23) 4067 135 No GCCGC CGGAG CGGAT GGAGG hum3D6 VH-B CCACC CACTC CAGGC CCTTG CCGGG GGCCT GGCGC ACCCA GGACA TGCCGTAGTT GGAGA AGGTG AAGCC GGAGG CGGCG CAGGA CAGGC GCAGG GAGCC GCCGG GCTGCACCAG (SEQ ID NO:24) 4068 142 Yes CTGGA GTGGG TGGCC TCCAT hum 3D6 VH-CCCGCT CCGGC GGCGG CCGCA CCTAC TACTC CGACA ACGTG AAGGG CCGCT TCACC ATCTCCCGCG ACAAC GCCAA GAACT CCCTG TACCT GCAGA TGAAC TCCCT GCGCG CCGAG GACACCG (SEQ ID NO:25) 4069 144 No ctgca aggat ccact caccG hum 3D6 VH-D GAGGACACGG TCACC AGGGT GCCCT GGCCC CAGTA GTCGG AGGAG CCGGA GTAGT GGTCG TAGCGCACGC AGTAG TACAG GGCGG TGTCC TCGGC GCGCA GGGAG TTCAT CTGCA GGTAC AGGG(SEQ ID NO:26) 4070 16 No GCCGC CGGAG CGGAT G hum 3D6 VH A + B (SEQ IDNO:27) back % A + T = 18.75 [3]; % C + G = 81.25 [13] Davis, Botstein,Roth Melting Temp C. 66.96 4071 20 Yes CTGGA GTGGG TGGCC TCCAT hum 3D6VH C + D (SEQ ID NO:28) forward % A + T = 35.00 [7]; % C + G = 65.00[13] Davis, Botstein, Roth Melting Temp C. 66.55 4072 19 Yes tcc gca agcttg ccg cca Hum 3D6 VL A + B c Forward (SEQ ID NO:29) % A + T = 31.58[6]; % C + G = 68.42 [13] Davis, Botstein, Roth Melting Temp C. 66.644073 29 No aat tct agg atc cac tca Hum 3D6 VL C + D cgC TTG ATC TC Back(SEQ ID NO:30) % A + T = 55.17 [16]; % C + G = 44.83 [13] Davis,Botstein, Roth Melting Temp C. 66.04 4074 23 Yes aca gaa agc ttg ccg ccaHum 3D6 VH A + B ccA TG Forward (SEQ ID NO:31) % A + T = 43.48 [10]; % C+ G = 56.52 [13] 66.33 4075 22 No ctg caa gga tcc act cac Hum 3D6 VH C+ D CGG A Back (SEQ ID NO:32) % A + T = 40.91 [9]; % C + G = 59.09 [13]66.40

The humanized light chain was assembled using PCR. DNA sequence analysisof greater than two dozen clones revealed scattered point mutations anddeletions throughout the VL region with respect to the expectedsequence. Analysis of the sequences indicated that clone 2.3 wasamenable to repair of 2 closely spaced single nucleotide deletions inthe amino-terminal region. Hence site directed mutagenesis was performedon clone pCRShum3D6v12.3 using oligonucleotides to introduce the 2deleted nucleotides, and repair of the point mutations was confirmed byDNA sequence analysis, and the VL insert was cloned into the light chainexpression vector pCMV-cK.

Assembly of humanized VH using PCR-based methods resulted in clones withgross deletions in the 5′ half of the sequence. Further efforts tooptimize the PCR conditions met with partial success. The clonesassembled via optimized PCR conditions still had 10-20 nt deletions inthe region mapping to the overlap of the A+B fragments. Consequently, analternate strategy was employed for VH assembly utilizing DNA polymerase(T4, Klenow, and Sequenase) mediated overlap extension, followed by T4DNA ligase to covalently join the overlapping ends. DNA sequenceanalysis of a subset of the clones resulting from VH assembly using thelatter approach revealed scattered point mutations and deletions amongthe clones. Analysis of over two dozen clones revealed essentially thesame pattern as illustrated for the clones. The similar results observedfollowing first pass assembly of VH and VL clones suggests the DNAsequence errors observed resulted from automated synthesizer errorsduring the synthesis of the long DNAs employed for the assembly.

Humanized VH clone 2.7 was selected for site-directedmutagenesis-mediated repair of the 3 nucleotide deletions it wasobserved to contain.

Example XIII Characterization of Humanized 3D6v2 Antibody

A second version of humanized 3D6 was created having each of thesubstitutions indicated for version 1, except for the D→Y substitutionat residue 1. Substitution at this residue was performed in version 1because the residue was identified as a CDR interacting residue.However, substitution deleted a residue which was rare for humanimmunoglobulins at that position. Hence, a version was created withoutthe substitution. Moreover, non-germline residues in the heavy chainframework regions were substituted with germline residues, namely,H74=S, H77=T and H89=V. Kabat numbering for the version 2 light andheavy chains, is the same as that depicted in Tables 13 and 14,respectively, except that residue 1 of the version 2 light chain is asp(D), residue 74 of the heavy chain is ser (S), residue 77 of the heavychain is thr (T) and residue 89 of the heavy chain is val (V). Thenucleotide sequence of humanized 3D6 version 1 light and heavy chainsare set forth as SEQ ID NOs: 34 and 36, respectively. The nucleotidesequence of humanized 3D6 version 2 light and heavy chains are set forthas SEQ ID NOs: 35 and 37, respectively.

Example IX Functional Testing of Humanized 3D6 Antibodies

Binding of humanized 3D6v1 to aggregated Aβ. Functional testing ofhumanized 3D6v1 was conducted using conditioned media from transientlytransfected COS cells. The cells were transfected with fully chimericantibody, a mixture of either chimeric heavy chain+humanized lightchain, or chimeric light chain+humanized heavy chain, and lastly, fullyhumanized antibody. The conditioned media was tested for binding toaggregated Aβ1-42 by ELISA assay. The humanized antibody showed goodactivity within experimental error, and displayed binding propertiesindistinguishable from the chimeric 3D6 reference sample. The resultsare shown in Table 16. TABLE 16 hu VH/ ChVH/ Hu VH/ ng/ml Chimeric ChVLHuVL HuVL 690 0.867 600 0.895 260 0.83 230 0.774 200 0.81 190 0.811 870.675 77 0.594 67 0.689 63 0.648 29 0.45 25 0.381 22 0.496 21 0.438 9.60.251 8.5 0.198 7.4 0.278 7 0.232 3.2 0.129 2.3 0.124

To compare the binding affinities of humanized 3D6v1 and 3D6v2antibodies, ELISA analysis was performed using aggregated Aβ as theantigen. The results show that both 3D6v1 (H1L1) and 3D6v2 (H2L2) havenearly identical Aβ binding properties (FIG. 5).

Replacement NET (rNET) analysis of h3D6v2. The rNET epitope map assayprovides information about the contribution of individual residueswithin the epitope to the overall binding activity of the antibody. rNETanalysis uses synthesized systematic single substituted peptide analogs.Binding of an antibody being tested is determined against native peptide(native antigen) and against 19 alternative “single substituted”peptides, each peptide being substituted at a first position with one of19 non-native amino acids for that position. A profile is generatedreflecting the effect of substitution at that position with the variousnon-native residues. Profiles are likewise generated at successivepositions along the antigenic peptide. The combined profile, or epitopemap, (reflecting substitution at each position with all 19 non-nativeresidues) can then be compared to a map similarly generated for a secondantibody. Substantially similar identical maps indicate that antibodiesbeing compared have the same or similar epitope specificity.

This analysis was performed for 3D6 and humanized 3D6, version 2.Antibodies were tested for binding against the native Aβ peptideDAEFRHDSGY (SEQ ID NO:33). Residues 1-8 were systematically substitutedwith each of the 19 non-native residues for that position. Maps weregenerated accordingly for 3D6 and h3D6v2. The results are presented intabular form in Table 17. TABLE 17 Aβ: replacement Net Epitope (rNET)mapping of wt3D6 and humanized 3D6 Wildtype Humanized 3D6 3D6Substitution [OD] [OD] Residue 1 = A 0.464 0.643 C 0.450 0.628 D 0.5770.692 E 0.576 0.700 F 0.034 0.062 G 0.569 0.738 H 0.054 0.117 I 0.0480.118 K 0.033 0.057 L 0.073 0.148 M 0.039 0.072 N 0.587 0.757 P 0.0690.144 Q 0.441 0.689 R 0.056 0.155 S 0.569 0.762 T 0.450 0.702 V 0.0570.190 W 0.031 0.070 Y 0.341 0.498 Residue 2 = A 0.548 0.698 C 0.5530.694 D 0.119 0.222 E 0.563 0.702 F 0.577 0.717 G 0.527 0.720 H 0.5340.741 I 0.522 0.722 K 0.548 0.722 L 0.482 0.705 M 0.535 0.705 N 0.5250.735 P 0.445 0.707 Q 0.567 0.756 R 0.562 0.719 S 0.587 0.705 T 0.5520.712 V 0.550 0.702 W 0.553 0.701 Y 0.547 0.704 Residue 3 = A 0.0380.061 C 0.222 0.410 D 0.019 0.027 E 0.542 0.689 F 0.034 0.060 G 0.0160.019 H 0.016 0.020 I 0.019 0.024 K 0.053 0.090 L 0.019 0.026 M 0.0190.027 N 0.024 0.032 P 0.017 0.020 Q 0.153 0.406 R 0.015 0.023 S 0.0160.021 T 0.015 0.019 V 0.016 0.021 W 0.149 0.304 Y 0.016 0.020 Residue 4= A 0.016 0.020 C 0.020 0.023 D 0.017 0.020 E 0.016 0.021 F 0.557 0.703G 0.016 0.020 H 0.470 0.723 I 0.119 0.360 K 0.015 0.018 L 0.559 0.716 M0.549 0.725 N 0.085 0.089 P 0.030 0.056 Q 0.065 0.110 R 0.016 0.019 S0.026 0.031 T 0.016 0.021 V 0.213 0.494 W 0.291 0.568 Y 0.529 0.730Residue 5 = A 0.275 0.435 C 0.359 0.635 D 0.080 0.163 E 0.115 0.187 F0.439 0.569 G 0.485 0.679 H 0.577 0.680 I 0.510 0.671 K 0.573 0.693 L0.517 0.691 M 0.418 0.611 N 0.476 0.655 P 0.093 0.198 Q 0.388 0.565 R0.613 0.702 S 0.487 0.633 T 0.530 0.639 V 0.493 0.562 W 0.393 0.461 Y0.278 0.230 Residue 6 = A 0.587 0.707 C 0.585 0.703 D 0.584 0.701 E0.579 0.702 F 0.586 0.704 G 0.592 0.709 H 0.596 0.688 I 0.602 0.708 K0.585 0.691 L 0.584 0.688 M 0.583 0.687 N 0.580 0.686 P 0.587 0.705 Q0.570 0.695 R 0.576 0.686 S 0.573 0.689 T 0.573 0.700 V 0.588 0.715 W0.576 0.696 Y 0.595 0.708 Residue 7 = A 0.580 0.688 C 0.559 0.676 D0.573 0.681 E 0.565 0.677 F 0.546 0.668 G 0.562 0.679 H 0.557 0.675 I0.552 0.681 K 0.565 0.685 L 0.566 0.701 M 0.562 0.697 N 0.573 0.688 P0.582 0.678 Q 0.563 0.679 R 0.551 0.677 S 0.563 0.674 T 0.560 0.685 V0.563 0.687 W 0.547 0.685 Y 0.560 0.682 Residue 8 = A 0.573 0.687 C0.583 0.700 D 0.586 0.697 E 0.601 0.701 F 0.586 0.687 G 0.569 0.681 H0.559 0.683 I 0.568 0.686 K 0.557 0.698 L 0.570 0.686 M 0.571 0.693 N0.573 0.700 P 0.574 0.694 Q 0.590 0.703 R 0.589 0.699 S 0.599 0.719 T0.586 0.689 V 0.578 0.688 W 0.567 0.687 Y 0.574 0.680

Notably, the profiles are virtually identical for 3D6 and h3D6v2 whenone looks at the substitutions at each position (i.e., the valuesfluctuate in an identical manner when comparing the data in column 1(3D6) versus column 2 (h3D6v2). These data demonstrate that thespecificity of h3D6v2 is preserved, as the h3D6v2 rNET epitope map isvirtually identical to m3D6 using both Aβ residues 1-4 and 5-8.

Immunohistochemistry on PDAPP brain sections demonstrates specificity ofh3D6v1 antibody. Humanized 3D6v1 antibody recognized Aβ in cryostatprepared brain sections from PDAPP mice. Humanized 3D6v1 and PK1614 bothbound to PDAPP plaques in the same dose response fashion, as measured bythe amount of fluorescence (quantitated in pixels) per slide versus theamount of antibody used to stain the tissue (FIG. 6). Identicalanti-human secondary antibodies were used in this experiment.Sectioning, staining, and image procedures were previously described. Inidentical experiments, image analysis of h3D6v2 staining on PDAPP and ADbrain sections revealed that h3D6v2 recognizes Aβ plaques in a similarmanner to 3D6v1 (e.g., highly decorated plaques).

Competitive binding analysis of h3D6. The ability of h3D6 antibodies v1and v2 to compete with murine 3D6 was measured by ELISA using abiotinylated 3D6 antibody. Competitive binding analysis revealed thath3D6v1, h3D6v2, and chimeric PK1614 can all compete with m3D6 to bind Aβ(FIG. 7). h3D6v1 and h3D6v2 were identical in their ability to competewith 3D6 to Aβ. The 10D5 antibody was used as a negative control, as ithas a different binding epitope than 3D6. BIAcore analysis also revealeda high affinity of h3D6v1 and h3D6v2 for Aβ (Table 18). TABLE 18Affinity Measurements of Aβ Antibodies Using BIAcore Technology Antibodyka1 (1/Ms) kd1 (1/s) Kd (nM) Mu 3D6 4.06E+05 3.57E−04 0.88 Chimeric 3D64.58E+05 3.86E−04 0.84 Hu 3D6v1 1.85E+05 3.82E−04 2.06 Hu 3D6v2 1.70E+053.78E−04 2.24

In comparison to 3D6, which has a Kd of 0.88 nM, both h3D6v1 and h3D6v2had about a 2 to 3 fold less binding affinity, measured at 2.06 nM and2.24 nM for h3D6v1 and h3D6v2, respectively. The ELISA competitivebinding assay revealed an approximate 6-fold less binding affinity forh3D6v1 and h3D6v2. Typically humanized antibodies lose about 3-4 fold inbinding affinity in comparison to their murine counterparts. Therefore,a loss of about 3 fold (average of ELISA and BIAcore results) for h3D6v1and h3D6v2 is within the accepted range.

Ex vivo assay using h3D6v2 antibody. The ability of h3D6v2 to stimulatemicroglial cells was tested through an ex vivo phagocytosis assay (FIG.8). h3D6v2 was as effective as chimeric 3D6 at inducing phagocytosis ofAβ aggregates from PDAPP mouse brain tissue. IgG was used as a negativecontrol in this experiment because it is incapable of binding Aβ andtherefore cannot induce phagocytosis.

In vivo brain localization of h3D6. ¹²⁵I labeled h3D6v2, m3D6, andantibody DAE13 were each IV-injected into 14 individual PDAPP mice inseparate experiments. Mice were sacrificed after Day 7 and perfused forfurther analysis. Their brain regions were dissected and measured for¹²⁵I activity in specific brain regions. Radiolabel activity in thebrain was compared with activity in serum samples. Results are set forthin Tables 19 and 20, for serum and brain regions, respectively. TABLE 19m3D6 DAE13 Hu3D6 30389.1 17463.9 40963.8 12171 13200.6 24202.2 3418.236284.7 12472.4 18678.9 421.3 33851.8 27241 19702 27187.3 26398.824855.8 29016.9 27924.8 29287.4 33830.7 12008.4 12733.1 26734.9 29487.827722.5 30144.5 25498.6 30460.7 35126.9 9652 23320.1 28414.8 24599.37119.1 16956.1 29240 28093.5 18190.7 11922.7 24659.7 25671.4 17443.126748.9

TABLE 20 m3D6 DAE13 Hu3D6 (H2L2) cere cort hipp cere cort hipp cere corthipp 1991.9 1201.1 4024 1277.5 2522.9 5711.9 2424.6 3759.4 11622 238.9746.1 2523 502.5 2123.5 6965.8 1509.8 2274.9 7018.2 645.9 603 1241.12325 3528.2 7801.6 500 2265.9 5316.3 1000 2508.2 4644.2 232.7 849.81891.9 2736.2 5703.7 10395.5 1266.9 3737.9 7975.8 891.6 2621 8245.21192.2 3188 10170 1422 2398.7 7731.1 1102.6 2087.5 7292.3 2269.4 3481.49621.6 1700.4 2154.4 7124.1 1650.6 3488.4 10284.8 1526.7 3028 8331.3542.5 812.4 2456.8 712.9 2318.5 6643.3 1538.1 4194.1 11244.8 1309 3010.58693.5 1172.9 1953.6 7363 1245.7 1699.4 6831.2 1372.2 997.5 2425.41067.9 3697.2 12280.7 2708.8 2789 7887.4 778.6 1291.9 5654.4 1952.22120.7 6412.7 2251.3 3897.5 11121.5 1199.3 1683.4 4887.3 1005.2 1852.55121.4 1529.6 1772.2 7986.9 1021.8 3234.5 8036.2 961.5 3382.9 8473.1644.1 1663.4 5056.5 742.1 1056.7 3405.2 852.3 1943.2 6717.4 1516.41620.6 9888 1273.7 1320.8 4262.6 997.5 3065.7 10213.1

The data show that h3D6v2 localized to the brain, and was particularlyconcentrated in the hippocampal region where Aβ is known to aggregate.Brain counts for m3D6 and DAE13 were comparable to h3D6v2. All threeantibodies were able to cross the blood barrier as demonstrated by Aβplaque binding in vivo.

Example X Cloning and Sequencing of the Mouse 10D5 Variable Regions

Cloning and Sequence Analysis of 10D5 VH. The VH and VL regions of 10D5from hybridoma cells were cloned by RT-PCR using 5′ RACE procedures. Thenucleotide sequence (SEQ ID NO:13) and deduced amino acid sequence (SEQID NO:14) derived from two independent cDNA clones encoding the presumed10D5 VL domain, are set forth in Table 21 and FIG. 9. The nucleotidesequence (SEQ ID NO:15) and deduced amino acid sequence (SEQ ID NO:16)derived from two independent cDNA clones encoding the presumed 10D5 VHdomain, are set forth in Table 22 and FIG. 10. The 10D5 VL and VHsequences meet the criteria for functional V regions in so far as theycontain a contiguous ORF from the initiator methionine to the C-region,and share conserved residues characteristic of immunoglobulin V regiongenes. TABLE 21 Mouse 10D5 VL DNA sequenceATGAAGTTGCCTGTTAGGCTGTTGGTACTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATTATACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAAGAAAGTGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGGAA (SEQ ID NO: 13)*Leader peptide underlined

TABLE 22 Mouse 10D5 VH DNA sequence.ATGGACAGGCTTACTTCCTCATTCCTGCTGCTGATTGTCCCTGCATATGTCCTGTCCCAGGCTACTCTGAAAGAGTCTGGCCCTGGAATATTGCAGTCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGAGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGGGATGATGACAAGCGCTATAACCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAGAAAGCAGGTATTCCTCAAGATCACCAGTGTGGACCCTGCAGATACTGCCACATACTACTGTGTTCGAAGGCCCATTACTCCGGTACTAGTCGATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 15)*Leader peptide underlined.

Example XI Efficacy of mAb 3D6 on Various Neuropathological Endpoints inPDAPP Mice

This Example describes the efficacy of murine mAb 3D6 on variousneuropathological endpoints. A comparison is made between passiveimmunization with 3D6 (at varying doses) and active immunization with anAβ peptide.

Immunizations

PDAPP mice were passively immunized with mAb 3D6 at three differentdoses, 10 mg/kg, 1 mg/kg and 10 mg/kg once a month (1×4). An unrelatedIgGγ2a antibody (TY 11/15) and PBS injections served as controls. Activeimmunization with Aβ peptide served as a comparison. Between 20 and 35animals were analyzed in each group.

The neuropathological endpoints assayed include amyloid burden andneuritic burden.

Amyloid Burden

The extent of the frontal cortex occupied by amyloid deposits wasdetermined by immunostaining with 3D6 followed by quantitative imageanalysis. The results of this analysis are shown in Table 6. Each of theimmunotherapies led to a significant reduction of amyloid burden.

Neuritic Burden

Neuritic burden following passive immunization with 3D6 was determinedin PDAPP mice by immunostaining of brain sections with anti-APP antibody8E5 followed by quantitative image analysis. Neuritic dystrophy isindicated by the appearance of dystrophic neurites (e.g., neurites witha globular appearance) located in the immediate vicinity of amyloidplaques. The results of this analysis are shown in Table 7. 3D6 (IgGβ2aisotype, recognizing Aβ1-5) did not significantly reduce neuritic burdenas compared to active immunization with Aβ peptide. Previously, it hadbeen observed that 10D5 (IgGγ1 isotype recognizing Aβ 3-7) was unable tosignificantly reduce neuritic burden. TABLE 23 Frontal Cortex AmyloidBurden PBS TY 11/15 3D6, 10 mg/kg 3D6, 1 mg/kg 3D6, 10 mg/kg/4 wks.Active N 31 30 29 31 32 24 Median (% AB) 15.182297 13.303288 0.8656712.286513 1.470956 2.162772 Range 0.160-31.961 0-61.706 0-7.0640.077-63.362 0-10.688 0-30.715 p Value (*M-W) .9425 ns ***.0001***<.0001 ***<.0001 ***.0004 % Change N/A 12% 94% 85% 90% 86%

TABLE 24 Frontal Cortex Neuritic Burden PBS TY 11/15 3D6, 10 mg/kg 3D6,1 mg/kg 3D6, 10 mg/kg/4 wks. Active N 31 30 29 31 32 24 Median (% NB)0.3946 0.3958 0.4681 0.3649 0.4228 0.2344 Range 0-1.3828 0-2.68000-1.3098 0-1.5760 0-1.8215 0-1.1942 p Value (*M − W) .8967 ns .9587 ns.6986 ns >.9999 ***.0381 % Change 0% 0% 7% 0% 41%

The characterization of various neuropathological endpoints in the PDAPPmouse model of Alzheimer's disease may assist the skilled artisan indesigning appropriate human therapeutic immunization protocols.

Example XII Prevention and Treatment of Human Subjects

A single-dose phase I trial is performed to determine safety in humans.A therapeutic agent is administered in increasing dosages to differentpatients starting from about 0.01 the level of presumed efficacy, andincreasing by a factor of three until a level of about 10 times theeffective mouse dosage is reached.

A phase II trial is performed to determine therapeutic efficacy.Patients with early to mid Alzheimer's Disease defined using Alzheimer'sdisease and Related Disorders Association (ADRDA) criteria for probableAD are selected. Suitable patients score in the 12-26 range on theMini-Mental State Exam (MMSE). Other selection criteria are thatpatients are likely to survive the duration of the study and lackcomplicating issues such as use of concomitant medications that mayinterfere. Baseline evaluations of patient function are made usingclassic psychometric measures, such as the MMSE, and the ADAS, which isa comprehensive scale for evaluating patients with Alzheimer's Diseasestatus and function. These psychometric scales provide a measure ofprogression of the Alzheimer's condition. Suitable qualitative lifescales can also be used to monitor treatment. Disease progression canalso be monitored by MRI. Blood profiles of patients can also bemonitored including assays of immunogen-specific antibodies and T-cellsresponses.

Following baseline measures, patients begin receiving treatment. Theyare randomized and treated with either therapeutic agent or placebo in ablinded fashion. Patients are monitored at least every six months.Efficacy is determined by a significant reduction in progression of atreatment group relative to a placebo group.

A second phase II trial is performed to evaluate conversion of patientsfrom non-Alzheimer's Disease early memory loss, sometimes referred to asage-associated memory impairment (AAMI) or mild cognitive impairment(MCI), to probable Alzheimer's disease as defined as by ADRDA criteria.Patients with high risk for conversion to Alzheimer's Disease areselected from a non-clinical population by screening referencepopulations for early signs of memory loss or other difficultiesassociated with pre-Alzheimer's symptomatology, a family history ofAlzheimer's Disease, genetic risk factors, age, sex, and other featuresfound to predict high-risk for Alzheimer's Disease. Baseline scores onsuitable metrics including the MMSE and the ADAS together with othermetrics designed to evaluate a more normal population are collected.These patient populations are divided into suitable groups with placebocomparison against dosing alternatives with the agent. These patientpopulations are followed at intervals of about six months, and theendpoint for each patient is whether or not he or she converts toprobable Alzheimer's Disease as defined by ADRDA criteria at the end ofthe observation.

General Materials and Methods

A. Preparation of Polyclonal and Monoclonal Aβ Antibodies

The anti-Aβ polyclonal antibody was prepared from blood collected fromtwo groups of animals. The first group consisted of 100 female SwissWebster mice, 6 to 8 weeks of age. They were immunized on days 0, 15,and 29 with 100 μg of AN1792 combined with CFA/IFA. A fourth injectionwas given on day 36 with one-half the dose of AN1792. Animals wereexsanguinated upon sacrifice at day 42, serum was prepared and the serawere pooled to create a total of 64 ml. The second group consisted of 24female mice isogenic with the PDAPP mice but nontransgenic for the humanAPP gene, 6 to 9 weeks of age. They were immunized on days 0, 14, 28 and56 with 100 μg of AN1792 combined with CFA/IFA. These animals were alsoexsanguinated upon sacrifice at day 63, serum was prepared and pooledfor a total of 14 ml. The two lots of sera were pooled. The antibodyfraction was purified using two sequential rounds of precipitation with50% saturated ammonium sulfate. The final precipitate was dialyzedagainst PBS and tested for endotoxin. The level of endotoxin was lessthan 1 EU/mg.

The anti-Aβ monoclonal antibodies were prepared from ascites fluid. Thefluid was first delipidated by the addition of concentrated sodiumdextran sulfate to ice-cold ascites fluid by stirring on ice to a reacha final concentration of 0.238%. Concentrated CaCl₂ was then added withstirring to reach a final concentration of 64 mM. This solution wascentrifuged at 10,000×g and the pellet was discarded. The supernatantwas stirred on ice with an equal volume of saturated ammonium sulfateadded dropwise. The solution was centrifuged again at 10,000×g and thesupernatant was discarded. The pellet was resuspended and dialyzedagainst 20 mM Tris-HCl, 0.4 M NaCl, pH 7.5. This fraction was applied toa Pharmacia FPLC Sepharose Q Column and eluted with a reverse gradientfrom 0.4 M to 0.275 M NaCl in 20 mM Tris-HCl, pH 7.5.

The antibody peak was identified by absorbance at 280 nm and appropriatefractions were pooled. The purified antibody preparation wascharacterized by measuring the protein concentration using the BCAmethod and the purity using SDS-PAGE. The pool was also tested forendotoxin. The level of endotoxin was less than 1 EU/mg. titers, titersless than 100 were arbitrarily assigned a titer value of 25.

B. Measurement of Antibody Titers

Mice were bled by making a small nick in the tail vein and collectingabout 200 μl of blood into a microfuge tube. Guinea pigs were bled byfirst shaving the back hock area and then using an 18 gauge needle tonick the metatarsal vein and collecting the blood into microfuge tubes.Blood was allowed to clot for one hr at room temperature (RT), vortexed,then centrifuged at 14,000×g for 10 min to separate the clot from theserum. Serum was then transferred to a clean microfuge tube and storedat 4° C. until titered.

Antibody titers were measured by ELISA. 96-well microtiter plates(Costar EIA plates) were coated with 100 μl of a solution containingeither 10 μg/ml either Aβ42 or SAPP or other antigens as noted in eachof the individual reports in Well Coating Buffer (0.1 M sodiumphosphate, pH 8.5, 0.1% sodium azide) and held overnight at RT. Thewells were aspirated and sera were added to the wells starting at a1/100 dilution in Specimen Diluent (0.014 M sodium phosphate, pH 7.4,0.15 M NaCl, 0.6% bovine serum albumin, 0.05% thimerosal). Seven serialdilutions of the samples were made directly in the plates in three-foldsteps to reach a final dilution of 1/218,700. The dilutions wereincubated in the coated-plate wells for one hr at RT. The plates werethen washed four times with PBS containing 0.05% Tween 20. The secondantibody, a goat anti-mouse Ig conjugated to horseradish peroxidase(obtained from Boehringer Mannheim), was added to the wells as 100 μl ofa 1/3000 dilution in Specimen Diluent and incubated for one hr at RT.Plates were again washed four times in PBS, Tween 20. To develop thechromogen, 100 μl of Slow TMB (3,3′,5,5′-tetramethyl benzidine obtainedfrom Pierce Chemicals) was added to each well and incubated for 15 minat RT. The reaction was stopped by the addition of 25 μl of 2 M H₂SO₄.The color intensity was then read on a Molecular Devices Vmax at (450nm-650 nm).

Titers were defined as the reciprocal of the dilution of serum givingone half the maximum OD. Maximal OD was generally taken from an initial1/100 dilution, except in cases with very high titers, in which case ahigher initial dilution was necessary to establish the maximal OD. Ifthe 50% point fell between two dilutions, a linear extrapolation wasmade to calculate the final titer. To calculate geometric mean antibodytiters, titers less than 100 were arbitrarily assigned a titer value of25.

C. Brain Tissue Preparation

After euthanasia, the brains were removed and one hemisphere wasprepared for immunohistochemical analysis, while three brain regions(hippocampus, cortex and cerebellum) were dissected from the otherhemisphere and used to measure the concentration of various Aβ proteinsand APP forms using specific ELISAs (Johnson-Wood et al., supra).

Tissues destined for ELISAs were homogenized in 10 volumes of ice-coldguanidine buffer (5.0 M guanidine-HCl, 50 mM Tris-HCl, pH 8.0). Thehomogenates were mixed by gentle agitation using an Adams Nutator(Fisher) for three to four hr at RT, then stored at −20° C. prior toquantitation of Aβ and APP. Previous experiments had shown that theanalytes were stable under this storage condition, and that synthetic Aβprotein (Bachem) could be quantitatively recovered when spiked intohomogenates of control brain tissue from mouse littermates (Johnson-Woodet al., supra).

D. Measurement of Aβ Levels

The brain homogenates were diluted 1:10 with ice cold Casein Diluent(0.25% casein, PBS, 0.05% sodium azide, 20 μg/ml aprotinin, 5 mM EDTA pH8.0, 10 μg/ml leupeptin) and then centrifuged at 16,000×g for 20 min at4° C. The synthetic Aβ protein standards (1-42 amino acids) and the APPstandards were prepared to include 0.5 M guanidine and 0.1% bovine serumalbumin (BSA) in the final composition. The “total” Aβ sandwich ELISAutilizes monoclonal antibody monoclonal antibody 266, specific for aminoacids 13-28 of Aβ (Seubert et al., supra), as the capture antibody, andbiotinylated monoclonal antibody 3D6, specific for amino acids 1-5 of Aβ(Johnson-Wood et al., supra), as the reporter antibody. The 3D6monoclonal antibody does not recognize secreted APP or fill-length APP,but detects only Aβ species with an amino-terminal aspartic acid. Thecell line producing the antibody 3D6 has the ATCC accession numberPTA-5130, having been deposited at the ATCC (American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209) onApr. 8, 2003. This assay has a lower limit of sensitivity of ˜50 ng/ml(11 nM) and shows no cross-reactivity to the endogenous murine Aβprotein at concentrations up to 1 ng/ml (Johnson-Wood et al., supra).

The Aβ1-42 specific sandwich ELISA employs mAβ 21F12, specific for aminoacids 33-42 of Aβ (Johnson-Wood, et al. supra), as the capture antibody.Biotinylated mAβ 3D6 is also the reporter antibody in this assay whichhas a lower limit of sensitivity of about 125 μg/ml (28 μM, Johnson-Woodet al., supra). For the Aβ ELISAs, 100 μl of either mAβ 266 (at 10μg/ml) or mAβ 21F12 at (5 μg/ml) was coated into the wells of 96-wellimmunoassay plates (Costar) by overnight incubation at RT. The solutionwas removed by aspiration and the wells were blocked by the addition of200 μl of 0.25% human serum albumin in PBS buffer for at least 1 hr atRT. Blocking solution was removed and the plates were stored desiccatedat 4° C. until used. The plates were rehydrated with Wash Buffer[Tris-buffered saline (0.15 M NaCl, 0.01 M Tris-HCl, pH 7.5), plus 0.05%Tween 20] prior to use. The samples and standards were added intriplicate aliquots of 100 μl per well and then incubated overnight at4° C. The plates were washed at least three times with Wash Bufferbetween each step of the assay. The biotinylated mAβ 3D6, diluted to 0.5μg/ml in Casein Assay Buffer (0.25% casein, PBS, 0.05% Tween 20, pH7.4), was added and incubated in the wells for 1 hr at RT. Anavidin-horseradish peroxidase conjugate, (Avidin-HRP obtained fromVector, Burlingame, Calif.), diluted 1:4000 in Casein Assay Buffer, wasadded to the wells for 1 hr at RT. The calorimetric substrate, SlowTMB-ELISA (Pierce), was added and allowed to react for 15 minutes at RT,after which the enzymatic reaction was stopped by the addition of 25 μl2 N H2SO4. The reaction product was quantified using a Molecular DevicesVmax measuring the difference in absorbance at 450 nm and 650 nm.

E. Measurement of APP Levels

Two different APP assays were utilized. The first, designated APP-α/FL,recognizes both APP-alpha (α) and full-length (FL) forms of APP. Thesecond assay is specific for APP-α. The APP-α/FL assay recognizessecreted APP including the first 12 amino acids of Aβ. Since thereporter antibody (2H3) is not specific to the α-clip-site, occurringbetween amino acids 612-613 of APP⁶⁹⁵ (Esch et al., Science248:1122-1124 (1990)); this assay also recognizes full length APP(APP-FL). Preliminary experiments using immobilized APP antibodies tothe cytoplasmic tail of APP-FL to deplete brain homogenates of APP-FLsuggest that approximately 30-40% of the APP-α/FL APP is FL (data notshown). The capture antibody for both the APP-α/FL and APP-α assays ismAb 8E5, raised against amino acids 444 to 592 of the APP⁶⁹⁵ form (Gameset al., supra). The reporter mAb for the APP-α/FL assay is mAb 2H3,specific for amino acids 597-608 of APP⁶⁹⁵ (Johnson-Wood et al., supra)and the reporter antibody for the APP-α assay is a biotinylatedderivative of mAb 16H9, raised to amino acids 605 to 611 of APP. Thelower limit of sensitivity of the APP-αFL assay is about 11 ng/ml (150M) (Johnson-Wood et al.) and that of the APP-α specific assay is 22ng/ml (0.3 nM). For both APP assays, mAb 8E5 was coated onto the wellsof 96-well EIA plates as described above for mAb 266. Purified,recombinant secreted APP-α was used as the reference standard for theAPP-α assay and the APP-α/FL assay (Esch et al., supra). The brainhomogenate samples in 5 M guanidine were diluted 1:10 in ELISA SpecimenDiluent (0.014 M phosphate buffer, pH 7.4, 0.6% bovine serum albumin,0.05% thimerosal, 0.5 M NaCl, 0.1% NP40). They were then diluted 1:4 inSpecimen Diluent containing 0.5 M guanidine. Diluted homogenates werethen centrifuged at 16,000×g for 15 seconds at RT. The APP standards andsamples were added to the plate in duplicate aliquots and incubated for1.5 hr at RT. The biotinylated reporter antibody 2H3 or 16H9 wasincubated with samples for 1 hr at RT. Streptavidin-alkaline phosphatase(Boehringer Mannheim), diluted 1:1000 in specimen diluent, was incubatedin the wells for 1 hr at RT. The fluorescent substrate4-methyl-umbellipheryl-phosphate was added for a 30-min RT incubationand the plates were read on a Cytofluor™ 2350 fluorimeter (Millipore) at365 nm excitation and 450 nm emission.

F. Immunohistochemistry

Brains were fixed for three days at 40 C in 4% paraformaldehyde in PBSand then stored from one to seven days at 4° C. in 1% paraformaldehyde,PBS until sectioned. Forty-micron-thick coronal sections were cut on avibratome at RT and stored in cryoprotectant (30% glycerol, 30% ethyleneglycol in phosphate buffer) at −20° C. prior to immunohistochemicalprocessing. For each brain, six sections at the level of the dorsalhippocampus, each separated by consecutive 240 μm intervals, wereincubated overnight with one of the following antibodies: (1) abiotinylated anti-Aβ (mAb, 3D6, specific for human Aβ) diluted to aconcentration of 2 μg/ml in PBS and 1% horse serum; or (2) abiotinylated mAb specific for human APP, 8E5, diluted to a concentrationof 3 μg/ml in PBS and 1.0% horse serum; or (3) a mAb specific for glialfibrillary acidic protein (GFAP; Sigma Chemical Co.) diluted 1:500 with0.25% Triton X-100 and 1% horse serum, in Tris-buffered saline, pH 7.4(TBS); or (4) a mAb specific for CD11b, MAC-1 antigen, (ChemiconInternational) diluted 1:100 with 0.25% Triton X-100 and 1% rabbit serumin TBS; or (5) a mAb specific for MHC II antigen, (Pharmingen) diluted1:100 with 0.25% Triton X-100 and 1% rabbit serum in TBS; or (6) a ratmAb specific for CD 43 (Pharmingen) diluted 1:100 with 1% rabbit serumin PBS or (7) a rat mAb specific for CD 45RA (Pharmingen) diluted 1:100with 1% rabbit serum in PBS; or (8) a rat monoclonal Aβ specific for CD45RB (Pharmingen) diluted 1:100 with 1% rabbit serum in PBS; or (9) arat monoclonal Aβ specific for CD 45 (Pharmingen) diluted 1:100 with 1%rabbit serum in PBS; or (10) a biotinylated polyclonal hamster Aβspecific for CD3e (Pharmingen) diluted 1:100 with 1% rabbit serum in PBSor (11) a rat mAb specific for CD3 (Serotec) diluted 1:200 with 1%rabbit serum in PBS; or with (12) a solution of PBS lacking a primaryantibody containing 1% normal horse serum.

Sections reacted with antibody solutions listed in 1, 2 and 6-12 abovewere pretreated with 1.0% Triton X-100, 0.4% hydrogen peroxide in PBSfor 20 min at RT to block endogenous peroxidase. They were nextincubated overnight at 4° C. with primary antibody. Sections reactedwith 3D6 or 8E5 or CD3e mAbs were then reacted for one hr at RT with ahorseradish peroxidase-avidin-biotin-complex with kit components “A” and“B” diluted 1:75 in PBS (Vector Elite Standard Kit, Vector Labs,Burlingame, Calif.). Sections reacted with antibodies specific for CD45RA, CD 45RB, CD 45, CD3 and the PBS solution devoid of primaryantibody were incubated for 1 hour at RT with biotinylated anti-rat IgG(Vector) diluted 1:75 in PBS or biotinylated anti-mouse IgG (Vector)diluted 1:75 in PBS, respectively. Sections were then reacted for one hrat RT with a horseradish peroxidase-avidin-biotin-complex with kitcomponents “A” and “B” diluted 1:75 in PBS (Vector Elite Standard Kit,Vector Labs, Burlingame, Calif.).

Sections were developed in 0.01% hydrogen peroxide, 0.05%3,3′-diaminobenzidine (DAB) at RT. Sections destined for incubation withthe GFAP-, MAC-1- AND MHC II-specific antibodies were pretreated with0.6% hydrogen peroxide at RT to block endogenous peroxidase thenincubated overnight with the primary antibody at 4° C. Sections reactedwith the GFAP antibody were incubated for 1 hr at RT with biotinylatedanti-mouse IgG made in horse (Vector Laboratories; Vectastain Elite ABCKit) diluted 1:200 with TBS. The sections were next reacted for one hrwith an avidin-biotin-peroxidase complex (Vector Laboratories;Vectastain Elite ABC Kit) diluted 1:1000 with TBS. Sections incubatedwith the MAC-1- or MHC II-specific monoclonal antibody as the primaryantibody were subsequently reacted for 1 hr at RT with biotinylatedanti-rat IgG made in rabbit diluted 1:200 with TBS, followed byincubation for one hr with avidin-biotin-peroxidase complex diluted1:1000 with TBS. Sections incubated with GFAP-, MAC-1- and MHCII-specific antibodies were then visualized by treatment at RT with0.05% DAB, 0.01% hydrogen peroxide, 0.04% nickel chloride, TBS for 4 and11 min, respectively.

Immunolabeled sections were mounted on glass slides (VWR, Superfrostslides), air dried overnight, dipped in Propar (Anatech) and overlaidwith coverslips using Permount (Fisher) as the mounting medium.

To counterstain Aβ plaques, a subset of the GFAP-positive sections weremounted on Superfrost slides and incubated in aqueous 1% Thioflavin S(Sigma) for 7 min following immunohistochemical processing. Sectionswere then dehydrated and cleared in Propar, then overlaid withcoverslips mounted with Permount.

G. Image Analysis

A Videometric 150 Image Analysis System (Oncor, Inc., Gaithersburg, Md.)linked to a Nikon Microphot-FX microscope through a CCD video camera anda Sony Trinitron monitor was used for quantification of theimmunoreactive slides. The image of the section was stored in a videobuffer and a color- and saturation-based threshold was determined toselect and calculate the total pixel area occupied by the immunolabeledstructures. For each section, the hippocampus was manually outlined andthe total pixel area occupied by the hippocampus was calculated. Thepercent amyloid burden was measured as: (the fraction of the hippocampalarea containing Aβ deposits immunoreactive with mAb 3D6)×100. Similarly,the percent neuritic burden was measured as: (the fraction of thehippocampal area containing dystrophic neurites reactive with monoclonalantibody 8E5)×100. The C-Imaging System (Compix, Inc., CranberryTownship, PA) operating the Simple 32 Software Application program waslinked to a Nikon Microphot-FX microscope through an Optronics cameraand used to quantitate the percentage of the retrospenial cortexoccupied by GFAP-positive astrocytes and MAC-1- and MHC II-positivemicroglia. The image of the immunoreacted section was stored in a videobuffer and a monochrome-based threshold was determined to select andcalculate the total pixel area occupied by immunolabeled cells. For eachsection, the retrosplenial cortex (RSC) was manually outlined and thetotal pixel area occupied by the RSC was calculated. The percentastrocytosis was defined as: (the fraction of RSC occupied byGFAP-reactive astrocytes) X 100. Similarly, percent microgliosis wasdefined as: (the fraction of the RSC occupied by MAC-1- or MHCII-reactive microglia) X 100. For all image analyses, six sections atthe level of the dorsal hippocampus, each separated by consecutive 240μm intervals, were quantitated for each animal. In all cases, thetreatment status of the animals was unknown to the observer.

Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.All publications and patent documents cited herein, as well as textappearing in the figures and sequence listing, are hereby incorporatedby reference in their entirety for all purposes to the same extent as ifeach were so individually denoted.

From the foregoing it will be apparent that the invention provides for anumber of uses. For example, the invention provides for the use of anyof the antibodies to Aβ described above in the treatment, prophylaxis ordiagnosis of amyloidogenic disease, or in the manufacture of amedicament or diagnostic composition for use in the same.

1. Humanized 10D5 antibody.
 2. A humanized antibody, or fragmentthereof, comprising a humanized light chain comprising three light chaincomplementarily determining regions (CDRs) from the mouse monoclonalantibody 10D5 and a light chain variable region framework sequence froma human immunoglobulin light chain; and a humanized heavy chaincomprising three heavy chain CDRs from the mouse monoclonal antibody10D5 and a heavy chain variable region framework sequence from a humanimmunoglobulin heavy chain; wherein the light chain CDRs have thefollowing amino acid sequences: light chain CDR1: Arg Ser Ser Gln AsnIle Ile His Ser Asn Gly Asn Thr Tyr Leu Glu (residues 24-39 of SEQ IDNO: 14) light chain CDR2: Lys Val Ser Asn Arg Phe Ser (residues 55-61 ofSEQ ID NO: 14) light chain CDR3: Phe Gln Gly Ser His Val Pro Leu Thr(residues 94-102 of SEQ ID NO: 14) and the heavy chain CDRS have thefollowing amino acid sequences: heavy chain CDR1: Thr Ser Gly Met GlyVal Ser (residues 31-37 of SEQ ID NO: 16) heavy chain CDR2: His Ile TyrTrp Asp Asp Asp Lys Arg Tyr Asn Pro Ser Leu Lys Ser (residues 52-67 ofSEQ ID NO: 16) and, heavy chain CDR3: Arg Pro Ile Thr Pro Val Leu ValAsp Ala Met Asp Tyr (residues 100-112 of SEQ ID NO: 16)


3. An antibody fragment obtainable by enzymatic cleavage of thehumanized antibody of any one of claims 1-2.
 4. An Fab or F(ab′)2fragment of any one of the humanized antibodies of claims 1-2.
 5. TheF(ab′)2 fragment of claim
 4. 6. The Fab fragment of claim
 4. 7. Thehumanized antibody or fragment of any one of claims 1-6, which is asingle chain antibody.
 8. The humanized antibody or fragment of any oneof claims 1-7 that is an IgG1 immunoglobulin isotype.
 9. The humanizedantibody or fragment of any one of claims 1-8, wherein the antibody orfragment thereof is produced in a host cell selected from the groupconsisting of a myeloma cell and a chinese hamster ovary (CHO) cell. 10.A polynucleotide compound, comprising a sequence coding for the lightchain or the heavy chain of the humanized antibody of any one of claims1-9, or a fragment thereof.
 11. A polynucleotide sequence, which whenexpressed in a suitable host cell, yields an antibody of any one ofclaims 1-9.
 12. An expression vector for expressing the antibody of anyone of claims 1-9 comprising the polynucleotide sequence of any one ofclaims 10-11.
 13. A cell transfected with the expression vector of claim12.
 14. A cell transfected with two expression vectors of claim 12,wherein a first vector comprises the polynucleotide sequence coding forthe light chain and a second vector comprises the sequence coding forthe heavy chain.
 15. A cell that is capable of expressing the humanizedantibody or fragment of any one of claims 1-9.
 16. The cell of any oneof claims 13-15, wherein the cell is selected from the group consistingof a myeloma cell and a chinese hamster ovary (CHO) cell.
 17. Apharmaceutical composition comprising the humanized antibody or fragmentof any one of claims 1-9, and a pharmaceutically acceptable excipient.18. A method of treating Down's syndrome or clinical or pre-clinicalAlzheimer's disease in a human subject, comprising administering to thehuman subject an effective amount of a humanized antibody or fragment ofany one of claims 1-9.
 19. A method to inhibit the formation of Aβplaque in the brain of a human subject, comprising administering to thehuman subject an effective amount of the humanized antibody or fragmentof any one of claims 1-9.
 20. A method to reduce Aβ plaque in the brainof a human subject, comprising administering to the human subject aneffective amount of a humanized antibody or fragment of any one ofclaims 1-9.
 21. The method of either of claims 19-20, wherein thesubject is diagnosed with clinical or pre-clinical Alzheimer's diseaseor Down's syndrome.
 22. The method of any one of claims 19-20, whereinthe subject is not diagnosed with clinical or pre-clinical Alzheimer'sdisease or Down's syndrome.
 23. Use of the humanized antibody or afragment thereof according to any one of claims 1-9 for the manufactureof a medicament, including prolonged expression of recombinant sequencesof the antibody or antibody fragment in human tissues, for treatingclinical or pre-clinical Alzheimer's disease or Down's syndrome.
 24. Useof the humanized antibody or fragment of any one of claims 1-9 for themanufacture of a medicament for treating Alzheimer's disease.
 25. Atleast one isolated mammalian amyloid antibody, comprising either (i) atleast two of the heavy chain complementarity determining regions (CDR)amino acid sequences of at least one of SEQ ID NOS:67-68; or (ii) atleast two of the light chain CDR amino acids sequences of at least oneof SEQ ID NOS:65.
 26. At least one isolated mammalian amyloid antibody,comprising at least one heavy chain or light chain CDR having the aminoacid sequence of at least one of SEQ ID NOS:67-68 and
 65. 27. At leastone isolated mammalian amyloid antibody that binds to the same region ofan amyloid polypeptide as an antibody comprising at least one heavychain or light chain CDR having the amino acid sequence of at least oneof SEQ ID NOS:67-68 and
 65. 28. At least one isolated mammalian amyloidantibody, comprising at least one human CDR, wherein said antibodyspecifically binds at least one epitope selected from amino acids 2-7,3-8, 33-42, or 34-40 of SEQ ID NO:70.
 29. At least one isolatedmammalian amyloid antibody, comprising at least one human CDR, whereinsaid antibody specifically binds at least one epitope comprising atleast 1-3, to the entire amino acid sequence of SEQ ID NO:70.
 30. Anamyloid antibody according to any of claims 25-27, wherein said antibodybinds amyloid with an affinity of at least one selected from at least10-9 M and at least 10-10 M.
 31. An amyloid antibody according to any ofclaims 25-27, wherein said antibody substantially modulates at least oneactivity of at least one amyloid polypeptide.
 32. An isolated nucleicacid encoding at least one isolated mammalian amyloid antibody accordingto any of claims 25-27 and having at least one human CDR of SEQ IDNOS:15 and
 13. 33. An isolated nucleic acid vector comprising anisolated nucleic acid according to claim
 32. 34. A prokaryotic oreukaryotic host cell comprising an isolated nucleic acid according toclaim
 32. 35. A host cell according to claim 34, wherein said host cellis at least one selected from COS-1, COS-7, HEK293, CHO, HeLa ormyeloma, cells, or any derivative, immortalized or transformed cellthereof.
 36. A method for producing at least one amyloid antibody,comprising translating a nucleic acid according to claim 32 underconditions in vitro, in vivo or in situ, such that the amyloid antibodyis expressed in detectable or recoverable amounts.
 37. A compositioncomprising at least one isolated mammalian amyloid antibody according toany of claims 25-27 having at least one human CDR, wherein said antibodyspecifically binds at least one epitope comprising at least 1-3, to theentire amino acid sequence of SEQ ID NO:70, and at least onepharmaceutically acceptable carrier or diluent.
 38. A compositionaccording to claim 37, further comprising at least one at least onecompound or polypeptide selected from at least one of a detectable labelor reporter, a drug, an immunomodulation agent, an opthalmic, otic ornasal drug, a topical drug, a nutritional drug, a cytokine, and acytokine antagonist.
 39. An anti-idiotype antibody or fragment thatspecifically binds at least one amyloid antibody according to any ofclaim 25-27.
 40. A method for diagnosing or treating an amyloid relatedcondition in a cell, tissue, organ or animal, comprising (a) contactingor administering a composition comprising an effective amount of atleast one antibody according to any of claim 25-27, with, or to, saidcell, tissue, organ or animal.
 41. A method according to claim 40,wherein said effective amount is 0.001-50 mg/kilogram of said cells,tissue, organ or animal.
 42. A method according to claim 40, whereinsaid contacting or said administrating is by at least one mode selectedfrom parenteral, subcutaneous, intramuscular, intravenous,intraperitoneal, rectal, intranasal, intrachranial, transdermal, andpulmonary.
 43. A method according to claim 40, further comprisingadministering, prior, concurrently or after said (a) contacting oradministering, at least one composition comprising an effective amountof at least one compound or polypeptide selected from at least one of adetectable label or reporter, a drug, an immunomodulation agent, anophthalmic, otic or nasal drug, a topical drug, a nutritional drug, acytokine, and a cytokine antagonist.
 44. A medical device, comprising atleast one amyloid antibody according to any of claim 25-27, wherein saiddevice is suitable to contacting or administering said at least oneamyloid antibody by at least one mode selected from parenteral,subcutaneous, intramuscular, intravenous, intracranial, intraperitoneal,bolus, rectal, intranasal, and transdermal.
 45. A kit for humanpharmaceutical or diagnostic use, comprising packaging material and acontainer comprising at least one amyloid antibody according to any ofclaim 25-27.
 46. The kit of claim 45, wherein said container is acomponent of a parenteral, subcutaneous, intramuscular, intravenous,intracranial, intraperitoneal, bolus, rectal, intranasal, or transdermaldelivery device or system.
 47. A method for producing at least oneisolated mammalian amyloid antibody according to any of claim 25-27,comprising providing a host cell or transgenic animal or eukaryotic cellcapable of expressing in recoverable amounts said antibody.
 48. At leastone amyloid antibody produced by a method according to claim 47.